ORIGINAL RESEARCH

published: 16 February 2022

doi: 10.3389/fmicb.2022.749874

Metaexoproteomics Reveals
Microbial Behavior in the Ocean’s
Interior
Zhang-Xian Xie 1,2,3† , Yan-Bin He 4† , Shu-Feng Zhang 1 , Lin Lin 1,3 , Ming-Hua Wang 1 and
Da-Zhi Wang 1,3*
1
State Key Laboratory of Marine Environmental Science/College of the Environment and Ecology, Xiamen University,
Xiamen, China, 2 College of Ocean and Earth Sciences, Xiamen University, Xiamen, China, 3 Southern Marine Science
and Engineering Guangdong Laboratory (Zhuhai), Sun Yat-sen University, Zhuhai, China, 4 BGI-Shenzhen, Shenzhen, China

The proteins present in the extracellular environment of cells, named the “exoproteome,”
are critical for microbial survival, growth, and interaction with their surroundings.
However, little is known about microbial exoproteomes in natural marine environments.
Edited by: Here, we used a metaproteomic approach to characterize the exoprotein profiles (10
Hongbin Liu,
Hong Kong University of Science
kDa-0.2 µm) throughout a water column in the South China Sea. Viruses, together
and Technology, Hong Kong SAR, with Alpha- and Gammaproteobacteria were the predominant contributors. However,
China
the exoprotein-producing microbial communities varied with depth: SAR11 in the
Reviewed by:
shallow waters, Pseudomonadales and Nitrososphaeria in the mesopelagic layer,
Benjamin Tully,
University of Southern California, and Alteromonadales, Rhizobiales, and Betaproteobacteria in the bathypelagic layer.
United States Besides viral and unknown proteins, diverse transporters contributed substantially
Daniele De Corte,
Japan Agency for Marine-Earth
to the exoproteomes and varied vertically in their microbial origins, but presented
Science and Technology (JAMSTEC), similar patterns in their predicted substrate identities throughout the water
Japan
column. Other microbial metabolic processes subject to vertical zonation included
*Correspondence:
proteolysis, the oxidation of ammonia, nitrite and carbon monoxide, C1 metabolism,
Da-Zhi Wang
dzwang@xmu.edu.cn and the degradation of sulfur-containing dissolved organic matter (DOM). Our
† These authors have contributed metaexoproteomic study provides insights into the depth-variable trends in the in situ
equally to this work ecological traits of the marine microbial community hidden in the non-cellular world,
Specialty section:
including nutrient cycling, niche partitioning and DOM remineralization.
This article was submitted to
Keywords: metaexoproteomics, exoprotein, microbial community, metabolic function, ocean water column
Aquatic Microbiology,
a section of the journal
Frontiers in Microbiology
INTRODUCTION
Received: 30 July 2021
Accepted: 10 January 2022
Exoproteins are present in the extracellular environment in close proximity to specific biological
Published: 16 February 2022
systems after their release by cell secretion, lysis, or leakage (Christie-Oleza et al., 2015). These
Citation: proteins are collectively designated the “exoproteome,” which is an indicator of the physiological
Xie Z-X, He Y-B, Zhang S-F, Lin L,
state of the cells under specific conditions, and can provide insights into the interactions
Wang M-H and Wang D-Z (2022)
Metaexoproteomics Reveals Microbial
between microbial cells and their environments, including nutrient uptake and cell competition
Behavior in the Ocean’s Interior. (Armengaud et al., 2012). Moreover, the significant contribution of exoproteins to the total
Front. Microbiol. 13:749874. extracellular enzymatic activity in marine ecosystem has been observed (Baltar et al., 2013,
doi: 10.3389/fmicb.2022.749874 2017). Cell-free active enzymes have also been documented in epipelagic and bathypelagic

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Xie et al. Metaexoproteomics of Ocean Water Column

waters (Baltar, 2018). Therefore, exoproteome analyses, i.e., toward to the disappearance of light, lower temperatures, higher
characterization of the composition, origin and function of pressures, and a lower carbon supply, which largely determine
exoproteins, are essential to fully understand the roles of the decline in biomass and the change in the composition of
microbes in natural ecosystems. the microbiome (DeLong et al., 2006). Despite their energy
Several in silico analyses have shown that 8–44% of the supply based on nitrification and sulfur metabolism as well
encoded proteomes of marine bacteria can be exported to the as diverse pathways of inorganic carbon fixation, microbes in
extracellular environment as theoretical exoproteomes (Evans the dark ocean are assumed to be primarily dependent on
et al., 2007; Christie-Oleza et al., 2012, 2015), indicating the heterotrophic metabolism of carbon exported from the
the significance of exoproteins in marine microbial ecology. sunlit layer (Swan et al., 2011; Pachiadaki et al., 2017; Acinas
However, our knowledge of the exoproteomes of marine et al., 2021). The vertical stratification of oceanic microbes has
bacteria is still limited because we have lacked the appropriate been verified in the cellular fraction with both metagenomics
methodology to study exoproteins. The shotgun proteomic and metatranscriptomics, e.g., the depth-variable metabolism
approach has been used to study the exoproteome of a of carbon, nitrogen and energy, and host-virus interactions
marine bacterium, Pseudoalteromonas tunicata, revealing the (DeLong et al., 2006; Shi et al., 2010). However, whether there
importance of iron transport and acquisition in this species is similar vertical zonation in the cell-free fraction of the
(Evans et al., 2007). Using a similar approach, exoproteomic water column remains to be examined. Here, we present a
studies of two strains within the marine Roseobacter clade metaexoproteomic analysis throughout the water column in the
show unsurprisingly abundant repeat-in-toxin (RTX) proteins, basin of the South China Sea (SCS), using a metaproteomic
which act as virulence factors in many pathogens (Christie-Oleza approach. We showed that the microbial contributors to and the
and Armengaud, 2010; Durighello et al., 2014). These studies functional categories of the vertical exoprotein profiles (10 kDa-
demonstrate the feasibility of examining exoprotein profiles with 0.2 µm) shifted from the sunlit zone to the dark deep ocean. The
this proteomic approach to investigate the ecological functions of findings of this study shed light on the ecologically significant
marine microbes. functions in the “non-cellular” world of the ocean.
Exoprotein profiles not only differ among microbial groups
across large phylogenetic distances, as shown in the studies
cited above, but also among very closely related microbes. MATERIALS AND METHODS
A comparative exoproteomic study of 12 Roseobacter strains
shows that the RTX proteins are only overrepresented in In situ Sampling
some strains (Christie-Oleza et al., 2012). Another comparative Using a rosette sampler equipped with a 12 L Niskin Go-Flo
exoproteomic study of eight Synechococcus strains reveals bottle, metaexoproteomic samples were collected from the DCM
obvious variations in the exoprotein profiles of strains with (75 m), upper mesopelagic layer (200 m), and bathypelagic
similar genetic backgrounds (Christie-Oleza et al., 2015). Both layer (3,000 m) at the SEATS station (18◦ N, 116◦ E) in the SCS
studies also demonstrate that the exoproteomes of the same in August 2012. To enrich the exoproteins for analysis, each
bacterial strains are growth-condition dependent. Therefore, sample was prepared from 100 L of seawater, and precautions
given the microbial diversity and complex environments were taken to avoid any possible contaminations during the
in natural marine habitats, the exoproteomes of marine experiment. In brief, sequential filtration with GF/F glass fiber
environments should be very dynamic. However, to our filters (0.7 µm pore size; Whatman) and Durapore membrane
knowledge, exoproteomic studies of marine microbes have been filters (0.2 µm pore size, Millipore) was performed gently at a
limited to two laboratory-cultured bacterial groups (Christie- filtration rate of less than 1 L/min to eliminate any remaining cells
Oleza et al., 2012, 2015), and little effort has been devoted once the seawater had arrived onboard. A final concentration
to examine natural marine exoproteomes, in the discipline of 0.01% (w/v) sodium dodecyl sulfate was added to increase
of “metaexoproteomics,” due to the lack of appropriate protein recovery and 5 mM of sodium azide was added to inhibit
methodology to study complex environmental samples. Though, microbial growth. A Pellicon 2 tangential flow filtration system
recent high-throughput analyses of metaproteomes in high equipped with a 10-kDa-cutoff regenerated cellulose membrane
molecular-weight dissolved organic matter (DOM) and marine package (Millipore) was used to concentrate the filtrate to a
viral concentrates from the euphotic zone have paved the volume of around 450 mL. The concentrates were stored at –80◦ C
way to the characterization of exoproteins in natural marine until a second concentration procedure was performed in the
environments (Dong et al., 2013; Xie et al., 2018). Based on laboratory to obtain the final metaexoproteomic samples (around
this method, a recent study is conducted on the exoproteomes 10 mL). Cell counting of the filtrates using flow cytometry showed
collected from marine epipelagic to bathypelagic waters, but it cell concentrations below the detection limit of flow cytometry
only focuses on two extracellular enzyme groups, i.e., peptidases (data not shown), indicating little contribution of intact cells to
and carbohydrate-active enzymes (CAZymes) (Zhao et al., 2020). the samples. It should be noted that concentrating large volumes
The sunlit zone is the most productive ocean region, and this of seawater was a time-consuming, laborious and expensive
ecosystem relies greatly on energy metabolism fueled by light, process, especially with deep seawater. Therefore, we collected
e.g., photosynthesis by phytoplankton and photoherterotrophy duplicate seawater samples from the DCM layer, but only one
by proteorhodopsin-containing bacteria (Ferrera et al., 2015). seawater sample each from the upper mesopelagic layer and the
Below the sunlit layer, the environment moves progressively bathypelagic layer due to the limitation of ship time.

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Xie et al. Metaexoproteomics of Ocean Water Column

Protein Extraction and Mass “combined non-redundant database” was created for the second
Spectrometry Analysis step of the target-decoy search using the three search engines
of MS-GF+, OMSSA, and X!Tandem integrated in the IPeak
The experimental procedures for protein precipitation,
software (Wen et al., 2015). The search parameters were set
preparation and final trypsin digestion for the MS analysis
as follows: precursor ion tolerance of 20 ppm; fragment ion
were conducted as described previously (Xie et al., 2018).
tolerance of 0.02 Da; trypsin as the proteolytic enzyme, allowing
Briefly, the exoproteins were precipitated with ice-cold 20%
for one missed cleavage; carbamidomethyl cysteine specified as a
trichloroacetic acid in acetone solution overnight at –20◦ C and
fixed modification and methionine oxidation (M) as a variable
were resuspended with rehydration buffer containing 7 M urea, 2
modification. In the second-step IPeak search, Percolator was
M thiourea, and 3-[(3-cholamidopropyl) dimethylammonium]-
used to re-score the peptide-spectrum matches from MS-GF+,
1-propane-sulfonate (4% w:v). Around 10–40 µg proteins of
OMSSA, and X!Tandem, and the results were combined based
each sample were obtained and subjected to trypsin digestion
on the FDRScore algorithm at a false discovery rate (FDR)
(12 h, 1:20 enzyme to protein) twice after reduction with
of 1%. Finally, a minimum of two peptides/proteins (at least
dithiothreitol and alkylation with iodoacetamide. The resulting
one unique peptide) was used to accept the protein matches as
peptide solution was fractionated with a Shimadzu LC-20AB
identifications with high confidence. The highest scoring protein
HPLC system equipped with a Ultremex strong cation exchange
in the group matching the same peptide was selected as the
column (4.6 × 250 mm, 5 µm particles; Phenomenex). The
representative protein.
elution gradient was set as follows: 0–10 min, 100% buffer A
A quantitative analysis was used to estimate the relative
[25 mM KH2 PO4 in 25% acetonitrile (ACN), pH 3.0]; 10–40 min,
abundances of the proteins (Pi ) with the equation:
5–35% buffer B (25 mM KH2 PO4 , 1 M KCl in 25% ACN, pH
3.0); 40–41 min, 35–80% buffer B; 41–44 min, 80% buffer B. Si
Li 
The peptide fractions were desalted with C18 columns and Pi = P  ∗100
n Sk
resuspended in buffer C [5% ACN, 0.1% formic acid (FA)]. k=1 Lk
The sample was loaded at 8 µL/min for 4 min, and analyzed
under a elution gradient [0–40 min, 2–35% buffer D (95% in which the unique spectral counts (Si ) are divided by the protein
ACN, 0.1% FA); 40–45 min, 35–80% buffer D; 45–49 min, length (Li ). The subscript i or k denotes a protein or a protein
80% buffer D] at 300 nL/min on a Shimadzu LC-20AD nano- group identity, and n is the total number of proteins or protein
HPLC in line with a Thermo Q Exactive mass spectrometer groups detected in a sample.
using data-dependent fragmentation with a dynamic exclusion All identified proteins were newly annotated using BLASTP
duration of 15 s. A normalized collision energy setting of 27.0 against the Clusters of Orthologous Groups of proteins (COG)
in the high-energy collision dissociation operating mode was database,1 the Kyoto Encyclopedia of Genes and Genomes
used to fragment selected peptides. Resolutions of 70,000 and (KEGG) database (version 81), and the National Center for
17,500 for the Orbitrap analyzer were used for the MS and Biotechnology Information Non-redundant Protein database
MS/MS scans, respectively, and the MS scans were set to between (NCBI-nr, 2017/9/24) with an E-value cutoff of 1e-5 and other
350 and 2,000 Da. default parameters. The taxonomic annotations were inferred
from taxonomic information on the hits in the NCBI-nr database.
Note that the taxonomic annotation of a bacterial protein was
Protein Identification and Bioinformatic modified to that of a putative viral protein if the protein function
Analysis was assigned to a viral structural protein.
The raw MS data for each sample were merged and formatted to
MGF files with Proteome Discoverer (version 1.3.0.339; Thermo
Fisher Scientific, San Jose, CA). Because we did not create sample- RESULTS
specific metagenomes, the proteins were identified and quantified
with a two-step iterative strategy similar to the previously Overview of Exoproteomes
described MetaPro-IQ approach (Zhang et al., 2016). Briefly, A high-throughput shotgun metaproteomic approach was used
more than 15 million protein-coding sequences were predicted to investigate exoprotein profiles collected from the deep
from the vertical metagenomes of the SCS (sizes between 0.2 chlorophyll maximum (DCM, 75 m), the upper mesopelagic
and 200 µm, and depths of 5 m, DCM, 200, 750, and 3,000 layer (200 m), and the bathypelagic layer (3,000 m) in an
m) in a previous study (Chen et al., 2021), and combined with oligotrophic basin of the SCS (Figure 1A). The tandem mass
the global Ocean Microbial Reference Gene Catalog (OM-RGC, spectrometry (MS/MS) datasets generated were screened against
more than 40 million sequences) of marine viruses, prokaryotes, a custom database containing environmental sequences from
and picoeukaryotes (Sunagawa et al., 2015) to construct a non- vertical metagenomes of the SCS (including one water column
redundant target-only database. The Mascot search engine (ver. at the same site) and the currently largest marine gene reservoir
2.3.0; Matrix Science, London, United Kingdom) was used (OM-RGC) with a two-step iterative strategy. They generated
in the first-step search, and all matched protein sequences 36,367 unique peptides matching 65,251 unique spectra. With a
were extracted as the sample-specific database without any stringent false discovery rate (FDR) cutoff of 1% and at least two
criteria. This reduced database was combined with the reverse
decoy database and duplicate sequences were removed, thus a 1
ftp.ncbi.nih.gov/pub/COG/COG2014/data

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Xie et al. Metaexoproteomics of Ocean Water Column

matched peptides, 3,354 exoproteins, accounting for 60% of the assigned to eukaryotic phytoplankton, such as Haptophyceae
protein abundance, were repeatedly detected in two biological (0.3–1.2%), Pelagophyceae (0.3–1.2%), and Prasinophyceae (0.2–
replicates of the DCM sample (DCM_1 and DCM_2), indicating 0.8%). Archaea contributed the least proteins (2.3–7.6%) to the
high reproducibility (Figure 1B). Finally, 6,037, 6,353, 6,069, and exoproteomes, and Nitrososphaeria [a reclassified class in GTDB
1,440 exoproteins were identified in four samples, with a total database for the NCBI phylum Thaumarchaeota (Rinke et al.,
output of 14,517 non-redundant exoproteins (Figure 1C and 2021), 1.0–6.3%] dominated this superkingdom.
Supplementary Data Sheets 1–5). Among these non-redundant The distribution patterns of microbial contributors to
exoproteins, 1.8–9.8% occurred in at least two depth layers, the exoproteomes varied throughout the water column. The
whereas only 1.0% was detected at all depths, indicating the protein abundances of most microbial groups declined with
distinctness of the exoproteomes in each water layer (Figure 1C). increasing depth. However, proteins from Pseudomonadales
(9.1%), Nitrososphaeria (6.3%), Candidatus Thioglobus (2.6%),
Taxonomic Characterization of Actinobacteria (2.6%), Nitrospinae (2.3%), Deltaproteobacteria
Exoproteomes (1.1%), Chloroflexi (1.0%), Nitrospirae (1.0%), and Chromatiales
The natural exoproteomes in the ocean contained exoproteins (0.9%) were the most abundant at 200 m, whereas those from
from all superkingdoms, although there was a small fraction Alteromonadales (20.2%), Rhizobiales (8.6%), Betaproteobacteria
of taxonomically unclassified proteins (Figure 2). Bacterial (7.4%), Oceanospirillales (2.2%), and Sphingomonadales (1.3%)
proteins dominated all the exoproteomes, and their relative were particularly abundant at 3,000 m.
abundances increased from 44% at the DCM to 69% in the dark
bathypelagic layer. Most of them originated from Proteobacteria Functional Characterization of
(30–58%), especially Alpha- and Gammaproteobacteria. Their Exoproteomes
descent taxonomic groups, including Pelagibacterales (SAR11), Virus-associated proteins were one of the major components
Rhizobiales, Alteromonadales, Pseudomonadales, and the FCB of the exoproteomes throughout the water column, although
(Fibrobacteres, Chlorobi, and Bacteroidetes) group, contributed their abundance decreased from 41% at the DCM to 12% at
the most proteins to the exoproteomes. Viruses contributed 200 m and 9% at 3,000 m. Of the virus-associated proteins, 48–
similar amounts of protein (37–41%) as bacteria at the DCM, 70% were viral structural components, such as capsid or tail
but the abundance of viral proteins decreased dramatically to proteins. The remaining virus-associated proteins were largely
12% at 200 m and to 9% at 3,000 m. Except for the large functionally uncharacterized (28–45%), with the exception of
amount of taxonomically unclassified proteins and putative several virus-encoded auxiliary metabolic proteins (AMPs),
viral proteins, most of the virus-associated proteins originated including the photosystem II D1 and D2 proteins, chaperonin
from the viral families Siphoviridae (1.1–4.2%), Myoviridae (1.0– GroES, and transporters of iron complexes and amino acids
3.5%), Podoviridae (0.02–2.1%), and Phycodnaviridae (0.07– (Supplementary Table 1).
2.2%). The abundances of eukaryotic proteins were similar The non-viral proteins included cellular proteins with diverse
among layers (4.7–8.9%), and the majority of them were functional categories (35–71%) and functionally unknown

FIGURE 1 | Numbers of proteins in the exoproteomes from the water layers at the deep chlorophyll maximum (DCM), 200 and 3,000 m at SEATS station in the
South China Sea (SCS). The map (A) shows the position of sampling site. Venn diagrams show the number and relative abundance of exoproteomes between two
biological replicates from the DCM (B) and between different water layers (C). In Venn diagrams, numbers without and in brackets show the proteins counts of each
part and their proportions of total relative abundance of four exoproteomes, respectively. Exoproteome from the DCM shown in (C) include all the non-redundant
proteins identified in the two biological replicates.

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Xie et al. Metaexoproteomics of Ocean Water Column

FIGURE 2 | Distribution of vertical exoprotein profiles in the SCS in terms of their microbial origins. Data are represented as percentage of total relative abundance of
each exoproteome.

proteins (17–24%) (Figure 3). At all depths, transporters relevant to posttranslational modification, protein turnover and
were the most abundant cellular proteins (15–34%), followed chaperones, carbohydrate metabolism, lipid metabolism, and cell
by moderately abundant proteins related to transcription motility were most abundant at 3,000 m.
and translation, energy metabolism, cytoskeleton, carbohydrate Many non-viral exoproteins were found to be related to
metabolism, posttranslational modification, protein turnover nutrient cycling and other important biogeochemical processes
and chaperones, nitrogen metabolism, amino acid metabolism, mediated by bacteria and archaea (Figures 4, 5). The distribution
defense mechanism, and cell motility. The remaining cellular of transporters in the exoproteomes throughout the water
proteins belonged to other functional categories and the column of the SCS provided the information on the substrate
abundance of each category did not exceed 2% in any uptake in exoprotein-producing microbial communities at
exoproteome sample. The distributions of different functional depths (Figure 4A). The substrate prediction was conducted
categories were depth dependent. Proteins associated with based on the KEGG annotation. With the exception of urea,
cytoskeleton, replication, recombination and repair, carbon nucleobases, and inorganic ions, almost all the known substrates
fixation, and photosynthesis were more abundant at the DCM associated with transporters were detected in each sample
than at the other two layers. However, proteins involved in (Figure 4A). Transporters indicating the utilization of fatty
transport, transcription and translation, nitrogen metabolism, acids, lipids, ammonia, and phosphate/phosphonate were more
amino acid metabolism, one-carbon metabolism, and coenzyme frequently detected in the dark ocean than at the DCM.
metabolism were most abundant at 200 m, and proteins Transporters of Fe3+ and iron complexes were abundantly

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Xie et al. Metaexoproteomics of Ocean Water Column

FIGURE 3 | Distribution of vertical exoprotein profiles in the SCS in terms of their functional categories. Data are represented as percentage of total relative
abundance of each exoproteome.

detected throughout the water column and were the dominant 3,000 m layer, 30, 17, 14, and 8% of eukaryotic exoproteins
transporters at 3,000 m, suggesting that iron was one of the were associated with cytoskeleton, protein chaperones,
most important nutrients recycled in the deep ocean. Vertical translation and ribosomal structure, and energy metabolism,
variations in transporters, classified by predicted substrates, were respectively. More than one third of these exoproteins were
apparent among the different prokaryotic taxonomic groups from eukaryotic phytoplankton, and included actin, tubulin,
(Figure 4B). The substrate uptake pattern of the exoprotein- heat shock proteins, ribosomal proteins, translation factors,
producing community at 200 m was rather similar to that and ATPases. Interestingly, most of these exoproteins from
at the DCM. One small difference was that several microbial eukaryotic phytoplankton, including the rubisco large subunit
groups, including Gammaproteobacteria, Betaproteobacteria, (rbcL), were also detected in the upper two layers, providing the
Deltaproteobacteria, Rhizobiales, and Archaea, consumed more first exoprotein evidence of biological pump in the ocean.
diverse substrates at 200 m. However, the patterns at the
DCM and at 3,000 m diverged in the microbial origins of
transporters (Figure 4B). For example, only a small fraction of the DISCUSSION
transporters from many microbial groups still remained at 3,000
m, and more substrates were associated with Alteromonadales
Vertical Changes in the
and Betaproteobacteria at 3,000 m than at the DCM. Moreover, Exoprotein-Producing Microbial
although exoproteins involved in proteolysis, nitrification, the Community
oxidation of carbon monoxide, C1 metabolism and sulfur Exoproteomic studies of laboratory-cultured marine bacteria,
metabolism were less abundant than transporters, variations such as Roseobacter and Synechococcus, have been reported
within these functional categories were also observed along the (Christie-Oleza and Armengaud, 2010; Christie-Oleza et al.,
water column (Figure 5). 2012, 2015; Durighello et al., 2014). However, little is known
Large amounts of non-viral exoproteins were from eukaryotes about the exoproteomics in natural marine environments. In
(4.7–8.9%) and were associated with diverse functions. In the this study, we used a metaproteomic approach to investigate the

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Xie et al. Metaexoproteomics of Ocean Water Column

FIGURE 4 | Distribution of transporters in the vertical exoproteomes from the SCS. (A) Transporter profiles classified by their predicted substrates. Data are
represented as percentage of total relative abundance of transporters detected in each sample. (B) Vertical profiles of transporters from different taxa. Bubble size
indicates the relative abundance of transporters summed by category.

exoproteomes throughout the water column. Diverse microbes associated with the diverse responses of different microbes to
contributed to the exoproteomes at each layer and the origins their environments.
of the exoproteins in the seawater were highly complex. The exoprotein-producing microbial community changed
Most of the microbial groups commonly found in the global from the sunlit zone to the dark deep ocean (Figure 2). To
ocean contributed to the marine exoproteomes. However, the some extent, the exoprotein abundance of many bacterial groups
relative abundances of the exoproteins varied in each layer along the water column showed similar trends with the vertical
in terms of their microbial contributors. This pattern can distribution of the bacterial population based on 16S rRNA genes
be partly explained by the genetic variations among different abundances in the SCS (Zhang et al., 2014). These microbial
microbial groups. For example, theoretically, 30% of the genes groups included SAR11, Rhizobiales, Sphingomonadales,
in the Roseobacter genome encode exoproteins, whereas this Alteromonadales, Rhodobacterales, Cyanobacteria, Nitrospinae,
increases to 40% in both cyanobacteria and SAR11 (Christie- and the FCB group. The highly abundant SAR11 exoproteins in
Oleza et al., 2012, 2015). The differences in biomass among each layer were consistent with the wide distribution of these
microbial groups was another important factor affecting the dominant bacteria in the global ocean (Morris et al., 2002).
exoprotein abundances. SAR11 and cyanobacteria, encoding Alteromonadales, Betaproteobacteria, and Oceanospirillales
similar proportions of exoproteome proteins, are both abundant are among the abundant bacterial groups in the deep-sea
at the DCM in the SCS (Zhang et al., 2014), which is microbial community (Eloe et al., 2011; Salazar et al., 2016)
consistent with their highly abundant exoproteins (Figure 2). and contributed strongly to the nonviral exoproteins at 3,000
The physiological status of cells or the contents released from m (Figure 2). However, the exoproteins of Pseudomonadales
dead cells can also change the exoproteomic outcomes (Christie- and Candidatus Thioglobus were especially abundant in the
Oleza et al., 2015), and the effect of these factors cannot be mesopelagic layer (Figure 2). Overall, the pattern of exoprotein
excluded. For example, the exoprotein abundance of hosts (e.g., abundances in the water column was related to the distributions
cyanobacteria and SAR11 bacteria) increased with that of their of the microbes in which they originated, somehow reflecting
associated viruses (Supplementary Figure 1), suggesting that microbial niche partitioning along the water column.
viral infection affects the exoprotein status. Moreover, microbes Eukaryotic phytoplankton contributed more than half the
associated with sinking particles might change physiologically eukaryotic exoproteins at the DCM, but declined to one-
or die in the deeper waters, consequently releasing their third in the other two layers, consistent with the vertical
cellular contents into the extracellular milieu. Overall, the distribution of phytoplankton in the ocean. The abundances of
exoprotein profiles of the microbial communities might be exoproteins derived from their predators, such as protozoa and

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Xie et al. Metaexoproteomics of Ocean Water Column

FIGURE 5 | Depth-variable pattern of selected exoproteins detected in the vertical exoproteomes in the SCS. Bubble size indicates the relative abundance of each
protein. Suox, sulfite oxidase; SsuD, alkanesulfonate monooxygenase; SseA, thiosulfate/3-mercaptopyruvate sulfurtransferase; SoxC, sulfane dehydrogenase
subunit SoxC; SBP56, 56-kDa selenium-binding protein; FccB, sulfide dehydrogenase; DmdC, 3-(methylthio) propanoyl-CoA dehydrogenase; AprA and AprB,
adenylylsulfate reductase subunits A and B; Tmm, trimethylamine monooxygenase; QhpA, quinohemoprotein amine dehydrogenase; MgsB and MgsC,
methylamine-glutamate N-methyltransferase subunits B and C; MauB, methylamine dehydrogenase heavy chain; Fhs, formate-tetrahydrofolate ligase; FdoH,
formate dehydrogenase iron-sulfur subunit; FdoG, formate dehydrogenase major subunit; Fae, 5,6,7,8-tetrahydromethanopterin hydro-lyase; CoxL, CoxM, and
CoxS, large, medium and small subunits of aerobic carbon-monoxide dehydrogenase; NxrA, NxrB, and NxrC, alpha, beta and gamma subunits of nitrite
oxidoreductase; NirK, nitrite reductase (NO-forming); AmoB, ammonia monooxygenase subunit B; ZmpB, zinc metalloprotease ZmpB; Vpr, minor extracellular serine
protease Vpr; Prc, carboxyl-terminal processing protease; PepN, aminopeptidase N; PfpI, protease I; PepD, putative serine protease PepD; HflK and HflC,
membrane protease subunits HflK and HflC; NprV, vibriolysin; Epr, minor extracellular protease Epr; DegQ, serine protease DegQ; DegP, serine protease Do; Cpt,
carboxypeptidase T; ColA, microbial collagenase; Clp, ATP-dependent Clp protease; Bpr, bacillopeptidase F; AprX, serine protease AprX; AprE, subtilisin; HslV,
ATP-dependent HslUV protease, peptidase subunit HslV.

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Xie et al. Metaexoproteomics of Ocean Water Column

Phycodnaviridae, also declined throughout the water column. the ocean (Sowell et al., 2009, 2011; Morris et al., 2010; Williams
This observation, together with the identification of cytoplasmic et al., 2012; Georges et al., 2014; Bergauer et al., 2018; Xie et al.,
proteins such as ribosomal proteins, translation factors and rbcL 2018). The predicted substrate identities of the transporters in
from eukaryotic phytoplankton, led us to postulate that the the vertical exoproteomes suggested that amino acids (including
lysis of phytoplankton cells by their predators was one of the branched-chain amino acids), polyamines, glycine betaine,
mechanisms by which the exoproteins of phytoplankton were oligopeptides, taurine, carbohydrates, and carboxylates were the
produced in marine environments. Several studies have reported major components of the labile dissolved organic nutrients
proteins from eukaryotic phytoplankton in size fractions both utilized by microbes throughout the water column in the SCS.
above and below 0.2 µm beneath the euphotic zone (Dong These results paralleled the results of a metaproteomic study in
et al., 2010; Wang et al., 2011; Moore et al., 2012). The the Atlantic Ocean that targeted the microbial community in the
observation of phytoplankton proteins in these studies could size fraction between 0.2 and 0.8 µm (Bergauer et al., 2018).
be attributed to preserved proteinaceous materials that have The predicted substrates of the transporters in
sunk from the euphotic zone, where phytoplankton usually the exoproteomes in each layer were linked to the
live. Protein solubilized from sinking particles or released microbial compositions of the communities (Figure 4B).
from live phytoplankton cells may result in the presence of These transporters mainly originated from lineages of
exoproteins from surface phytoplankton in aphotic waters. Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria,
Viable phytoplankton cells from the surface ocean have been the FCB group, Actinobacteria, and Archaea, consistent with
observed in the deep ocean (Lochte and Turley, 1988; Jiao the observations in other metaproteomic studies in the cellular
et al., 2013; Xu et al., 2018). Consistent with findings in the size fraction (Morris et al., 2010; Bergauer et al., 2018). SAR11,
deep Western Pacific Ocean (Xu et al., 2018), the presence of Rhodobacterales, Rhodospirillales, and Rhizobiales were strongly
eukaryotic exoproteins in the deep SCS supports the notion that representative Alphaproteobacteria in the shallow layers but
Haptophyceae, Pelagophyceae, and Prasinophyceae are the major contributed less in the deep sea; Candidatus Thioglobus,
eukaryotic phytoplankton contributing to the biological carbon Chromatiales, the FCB group, Actinobacteria, and Archaea
pump that fuels the carbon pool in the deep sea. contributed more-abundant transporters at 200 m than in the
Archaea are abundant below the euphotic zone (Karner et al., other two layers; whereas Alteromonadales, Pseudomonadales,
2001). In the present study, the higher abundance of archaeal and Betaproteobacteria contributed more transporters with
exoproteins at 200 m than at the DCM supports the idea that increasing depth. Therefore, it appears that divergent microbial
the dark ocean is the preferred habitat of archaea. In the ocean, groups drive nutrient cycling at different depths. As in a previous
Nitrososphaeria often dominate Euryarchaeota (Karner et al., study (Bergauer et al., 2018), the taxa associated with substrate-
2001; Salazar et al., 2016). Consistent with their greater biomass, classified transporters changed throughout the oceanic water
more exoproteins of Nitrososphaeria than Euryarchaeota were column (Figure 4B). Based on the transporter results for different
observed, especially at 200 m. Together, these data emphasize size fractions, both studies suggest the vertical stratification of
the ecological importance of Nitrososphaeria within the archaea, nutrient-based niche partitioning in the ocean.
particularly in the dark ocean. Overall, the transporters abundantly present in the vertical
exoproteomes lent support to the depth-dependent variation
in microbe-nutrient interactions in the ocean. Although the
Vertical Changes in Microbial Functions microbial origins of the transporters changed throughout the
Nutrient Uptake water column, the substrates utilized by the communities were
Among the functional cellular proteins detected, transporters largely labile DOM and changed only slightly with depth,
were the most abundant category in each layer (Figure 3). This which is similar to the results of a previous metaproteomic
is consistent with the high abundance of transporters in both study (Bergauer et al., 2018). This, together with the detection
the theoretical and experimental exoproteomes of laboratory- of exoproteins from surface phytoplankton at 3,000 m, also
cultured marine bacteria (Christie-Oleza and Armengaud, 2010; supported the hypothesis that deep sea microbes largely relied on
Armengaud et al., 2012; Christie-Oleza et al., 2012, 2015). the DOM solubilized from sinking particles, rather than on its
Most transporters detected in this study were the periplasmic local recalcitrant counterpart (Bergauer et al., 2018).
components of transport systems or outer-membrane receptor
proteins, which are exported to the extracellular space as the Proteolysis
prime determinants of the selectivity of substrate identities. Proteases and peptidases are a class of enzymes that break
The presence of these highly abundant transporters indicated proteins into smaller polypeptides or single amino acids. They
that one of the important ecological functions of the microbial are frequently detected in the exoproteomes of marine bacterial
exoproteome was closely associated with nutrient scavenging. isolates (Christie-Oleza et al., 2012, 2015) and the microbial
The highly abundant transporters in the deeper waters suggested community in the ocean (Zhao et al., 2020), and at least 20
that nutrient-associated interactions between microbes and their different proteases were identified in this study (Figure 5),
surroundings also occurred in the dark ocean. demonstrating that proteases are a common component of
The variability in transporters in terms of their predicted marine exoproteomes (Armengaud et al., 2012). The total
substrates and phylogenetic origins provides insights into the abundance of all proteases increased from epipelagic to
nutrient utilization dynamics of the microbial communities in bathypelagic waters in the SCS, and this pattern is consistent with

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Xie et al. Metaexoproteomics of Ocean Water Column

those in other ocean regions reported in a previous exoproteomic Nitrospirae, consistent with the genetic capacity of their cultured
study (Zhao et al., 2020), suggesting that protein recycling isolates and natural lineages for nitrite oxidation within the
became increasingly important as a pathway of carbon and nitrification process (Lücker et al., 2010; Lucker et al., 2013;
nitrogen supply for the microbial community in deeper waters. Koch et al., 2015; Pachiadaki et al., 2017). All subunits of NXR
However, different proteases varied in their vertical distributions, (NxrABC) showed the same vertical distribution pattern as Amo
including the extracellular proteases Vpr, Epr, Bpr, AprE/X, and and NirK. This close co-occurrence relationship might support
NprV. The Vpr protease increased in abundance with depth. Epr the previously proposed reciprocal feeding between AOA and
and Bpr were only detected at 200 m, where AprE/X and NprV NOB (Lücker et al., 2010; Pachiadaki et al., 2017). NXR proteins
were undetected, and AprE/X and NprV were most abundant at of Nitrospirae were only detected at 200 m, and were only half as
3,000 m. These five proteases act specifically in proteolysis, but abundant as their Nitrospinae counterparts, consistent with the
may also have other functions. For example, the secretion of Epr previous observation of their transcripts in the SCS (Zhang et al.,
is involved in bacterial swarming motility (Murudkar et al., 2006). 2020). The predominance of Nitrospinae over Nitrospirae in the
Vertical variations in proteases in terms of the enzyme species ocean in terms of exoprotein abundance is also consistent with
and their abundances probably reflect the changes in the protein observations based on a metagenomic or metatranscriptomic
substrates available for degradation and other protease-mediated recruitment analysis (Pachiadaki et al., 2017; Zhang et al., 2020).
processes at different depths. Taken together, these data indicated that the nitrification in the
water column of the SCS was achieved via ammonia oxidation by
Nitrification AOA and nitrite oxidation by NOB, and occurred most actively
A protein involved in the first step in nitrification, ammonia immediately below the euphotic zone.
monooxygenase subunit B (AmoB) from Nitrososphaeria was
detected in each layer. The failure to detect other subunits of Amo Carbon Monoxide Oxidation and C1 Metabolism
is consistent with the presence of abundant AmoB, as has been A wide range of subunits (CoxL, CoxM, and CoxS) of aerobic
observed in the proteomes of laboratory-cultured isolates and carbon monoxide dehydrogenase (CODH), which oxidizes CO
natural ammonia-oxidizing archaea (AOA) populations (Georges to CO2 , were detected from diverse heterotrophic bacteria,
et al., 2014; Hawley et al., 2014; Santoro et al., 2015; Kerou including SAR11, Bacteroidetes, Actinobacteria, and Chloroflexi,
et al., 2016; Qin et al., 2017). Moreover, protein homologues of indicating that they probably utilize CO. It has been estimated
the NO-forming nitrite reductase (NirK) family detected here that CoxL genes are as abundant as one per 14 bacterial cells,
all originated from Nitrososphaeria. In a previous model of based on a marine metagenomic library (Moran et al., 2004).
thaumarchaeal ammonia oxidation, NirK reduced nitrite to nitric Therefore, the frequent detection of CODH proteins throughout
oxide (NO), which was subsequently used for hydroxylamine the water column in this and other studies (Georges et al., 2014)
oxidation, although this was not supported by isotopic trace demonstrated the ecological significance of CO oxidation in the
experiments (Santoro et al., 2011; Kozlowski et al., 2016). Instead, ocean. It has been reported that up to 88% of CO is oxidized
in new models, NirK is proposed to act in the two-step oxidation by microbes, thus greatly reducing the entry of this greenhouse
of hydroxylamine to NO and then to nitrite (Carini et al., 2018). gas into the atmosphere (Tolli and Taylor, 2005). Therefore, the
Regardless of this debate, both Amo and NirK are believed to detection of CODH confirms that bacterial CO oxidation was an
be the core components of the ammonia oxidation machinery. important mechanism by which CO sinks in the SCS.
In the model, the predicted location of these proteins is the It has been experimentally demonstrated that SAR11 are
thaumarchaeal pseudoperiplasm (Carini et al., 2018), which is versatile in the utilization of diverse C1 or methylated
consistent with their detection in exoproteomes. NirK was more compounds, mainly for energy production rather than carbon
abundant than AmoB, consistent with the higher expression assimilation, in a process called “carboxidovory” (Sun et al.,
of NirK transcripts than AmoB transcripts (Lund et al., 2012). 2011). Proteins associated with the relevant metabolic processes
Similar to a previous study (Carini et al., 2018), only ammonium were identified from SAR11, including Tmm, which is involved
transporters from Nitrososphaeria were coexpressed with AmoB in the conversion of trimethylamine (TMA) to trimethylamine
and NirK in the present study, and were more abundantly N-oxide; MgsB and MgsC, which oxidizes methylamine to
represented in the aphotic zone, especially at 200 m (Figure 5). formaldehyde; Fae, which converts formaldehyde to formate
Ammonia oxidation was mainly associated with Nitrososphaeria via the tetrahydromethanopterin pathway; formamidase, which
and was more abundantly present below the bottom of euphotic degrades formamide to formate; and Fhs, which participates
zone in the SCS. In addition, an energy link between carbon in the assimilation of formate via the tetrahydrofolate pathway
fixation and ammonia oxidation in AOA has been proposed (Sun et al., 2011, 2019; Martinez-Gomez et al., 2013). This
(Williams et al., 2012; Georges et al., 2014; Santoro et al., 2015). supported the utilization of TMA, methylamine, formaldehyde,
Consistent with this, proteins of Nitrososphaeria involved in formamide, and formate by SAR11. Interestingly, no one-step
carbon fixation in the hydroxypropionate-hydroxybutylate cycle formate oxidation to CO2 occurred in SAR11 because no formate
were identified, including acetyl-/propionyl-CoA carboxylase, dehydrogenase was detected from SAR11, but only from other
4-hydroxybutyryl-CoA dehydratase, 3-hydroxypropionyl-CoA bacteria. In addition, methylamine was probably not oxidized
dehydratase, and methylmalonyl-CoA mutase. by SAR11 but degraded by Alteromonas at 3,000 m, because the
Nearly all nitrite oxidoreductases (NXRs) were associated with MauB and QhpA proteins in another methylamine oxidation
two groups of nitrite-oxidizing bacteria (NOB), Nitrospinae and pathway were detected. The vertical distribution of these proteins

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Xie et al. Metaexoproteomics of Ocean Water Column

indicated that the roles of SAR11 in the cycling of low-molecular- and the predominant contributors to the exoproteomes, such
weight DOM are depth-dependent. as viruses, Alpha- and Gammaproteobacteria, from the sunlit
zone to the deep dark ocean. The origin of exoproteins suggests
Sulfur Metabolism that these microbial groups make a significant contribution
Notably, a suite of proteins involved in sulfur metabolism to the oceanic DOM pool. However, the contribution
were detected, including DmdC, which acts in the pathway of of abundant taxa is depth-dependent, e.g., SAR11 in the
dimethylsulfoniopropionate demethylation; SBP56, which acts shallow waters, Pseudomonadales and Nitrososphaeria in
in the oxidation of methanethiol; and ssuD, which acts in the the mesopelagic layer, and Alteromonadales, Rhizobiales and
degradation of alkanesulfonate (Sun et al., 2016; Eyice et al., Betaproteobacteria in the bathypelagic layer. Furthermore,
2017). Except for DmdC, these proteins were not found in the we demonstrate the vertical zonation of exoprotein-linked
exoproteome at 3,000 m, indicating that the upper layers of the functions, including substrate transport, protein degradation,
SCS were the main regions of remineralization of these sulfur- nitrification, oxidation of C1 and methylated compounds, sulfur
containing DOMs. Interestingly, adenylylsulfate reductase and metabolism and host-virus interaction. Especially, microbial
sulfite oxidase predominantly originated from SAR11, and are players are depth-dependent despite of utilizing similar DOM
thought to detoxify sulfite, a byproduct of taurine degradation, substrates; different extracellular proteases are involved in
in this bacterial group (Williams et al., 2012). Similar to the protein recycling at different layers; nitrification by AOA
distribution of these proteins, SAR11 transporters that acted and NOB is more abundantly present below the euphotic
in taurine uptake were only found at the DCM and 200 m, zone, whereas degradation of sulfur-containing DOMs and
indicating that the SAR11-mediated remineralization of taurine host-virus interactions are more frequently observed in the
was probably confined to shallow waters. We also noticed that shallow layers. The depth-variable trends in these categories
exoprotein-linked sulfur metabolism in the deep SCS was not as of metabolic functions provide new insights into the dynamic
abundant as that in a recent metagenomic study of the global interactions between microbes and their surroundings in
dark ocean (Acinas et al., 2021). This might be attributable to terms of nutrient uptake, DOM remineralization, and niche
differences in the regulation of gene expression and the release partitioning in the ocean.
mechanisms of these proteins at different locations. Cell-free active enzymes have been documented in epipelagic
and bathypelagic waters, and their activities are regarded as the
Host-Virus Interaction
“gatekeepers” of biogeochemical cycles (Baltar, 2018). However,
The significant contribution of viral structural proteins to the
our current knowledge is mainly based on a few extracellular
metaexoproteomes throughout the water column demonstrated
enzymes. Our study indicates the presence of novel exoproteins
the presence of abundant viruses in the ocean and indicated
with potential connections to diverse biogeochemical processes
the potential interactions between viruses and their hosts. The
in the ocean. In future metaexoproteomic studies, more
presence of and variations of host-virus interaction at depths were
efforts should be directed toward the kinds of exoproteins
verified with the detection of viral AMPs in the exoproteomes
that are functionally active in the noncellular world, with
(Supplementary Table 1). AMPs are thought to be expressed to
the integration of multiple meta-omic approaches, such as
reprogram the host metabolism during viral infection (Lindell
metaexoproteomics and metabonomics, as well as in situ
et al., 2005). Therefore, despite that our metaproteomics-based
metabolic rate measurements. This will comprehensively
approach could only detected known viral AMPs in the public
improve our understanding of microbial functions and
databases, these AMPs present in the cell-free fraction probably
roles in the ocean.
derive from the recent release of these proteins from virus-
It should be pointed out that only one sample was collected in
infected cells. Furthermore, the prevalence and expression of the
both the meso- and bathypelagic layers owing to the limitation
viral psbA gene in the surface ocean (Sharon et al., 2007) and
of sampling time. Because the content of protein in particulate
the rapid sinking of virus-infected cells (Suttle, 2005) have been
organic carbon is very low, large volumes of seawater must be
reported. Our metaexoproteomic study not only detected viral
concentrated to obtain sufficient protein for proteomic analysis,
psbA proteins in both layers of the DCM and 200 m, but also
which is a time-consuming, laborious and expensive process. This
identified a psbD protein from Synechococcus phage in the 3,000
brings forward challenges for repeated sampling, especially in
m layer, suggesting that some cyanobacterial cells in the surface
deep waters. In oceanography, oceanographic consistency across
ocean were probably phage-infected and were lysed as they sank
the vertical depth structure is a good substitute for biological
to the deep ocean. Overall, our results demonstrate the depth-
replicates when validating results, because the fluid feature of
dependence of host-virus interactions and their roles in carbon
the ocean may allow different water masses to be captured, even
export in the ocean.
within a short time, leading to unreal variations in the microbial
community and other properties among replicates (Saito et al.,
CONCLUSION 2019). The strategy of sampling more sites can also improve
the results, as demonstrated in the previous study of oceanic
In this metaexoproteomic analysis, we investigate the natural exoproteomes focusing on CAZymes and peptidases (Zhao et al.,
exoprotein profiles throughout an oceanic water column. Our 2020). Overall, different sampling strategies should be adopted
study sheds light on the complex origins of the exoproteins according to actual field conditions, to avoid the analytical errors.

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Xie et al. Metaexoproteomics of Ocean Water Column

DATA AVAILABILITY STATEMENT ACKNOWLEDGMENTS

The data presented in the study are deposited in the We thank the captain and crew of the R/V DongFangHong 2 for
ProteomeXchange repository available here: www.ebi.ac.uk/ their assistance. We also thank International Science Editing
pride/archive/, accession number PXD020896. (http://www.internationalscienceediting.com) and Michelle
Michelsen from University of Exeter for editing this manuscript.

AUTHOR CONTRIBUTIONS
SUPPLEMENTARY MATERIAL
D-ZW and Z-XX conceived the study. Z-XX, S-FZ,
and M-HW collected the samples. Z-XX and Y-BH The Supplementary Material for this article can be found
analyzed the data. Z-XX and LL performed the online at: https://www.frontiersin.org/articles/10.3389/fmicb.
proteomic experiment. Z-XX, Y-BH, and D-ZW wrote the 2022.749874/full#supplementary-material
manuscript. All authors contributed to the discussion of
Supplementary Figure 1 | Scatter plot showing the abundance relationship
the results. between phage proteins and host exoproteins. Dots with different colors indicate
different host groups.

Supplementary Data Sheet 1 | Sequence information for exoproteins identified

FUNDING in the DCM_1 sample.

Supplementary Data Sheet 2 | Sequence information for exoproteins identified

This study was supported by the National Natural Science
in the DCM_2 sample.
Foundation of China through grants 41425021, the Ministry
of Science and Technology through grant 2016YFA0601202 Supplementary Data Sheet 3 | Sequence information for exoproteins identified
in the 200 m sample.
and 2015CB954003. D-ZW was also supported by the “Ten
Thousand Talents Program” for leading talents in science Supplementary Data Sheet 4 | Sequence information for exoproteins identified
and technological innovation. Z-XX gratefully acknowledge in the 3000 m sample.
financial support from the China Postdoctoral Science Supplementary Data Sheet 5 | Sequence information for non-redundant
Foundation (2019M652253). exoproteins identified in all samples.

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Xie, Z.-X., Chen, F., Zhang, S.-F., Wang, M.-H., Zhang, H., Kong, L.-F., absence of any commercial or financial relationships that could be construed as a
et al. (2018). Metaproteomics of marine viral concentrates reveals key viral potential conflict of interest.
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chlorophyll maximum of the South China Sea. Environ. Microbiol. 20, 477–491. Publisher’s Note: All claims expressed in this article are solely those of the authors
doi: 10.1111/1462-2920.13937 and do not necessarily represent those of their affiliated organizations, or those of
Xu, D., Sun, P., Zhang, Y., Li, R., Huang, B., Jiao, N., et al. (2018). Pigmented the publisher, the editors and the reviewers. Any product that may be evaluated in
microbial eukaryotes fuel the deep sea carbon pool in the tropical Western this article, or claim that may be made by its manufacturer, is not guaranteed or
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dark ocean. Proc. Natl. Acad. Sci. U.S.A. 117, 4823–4830. doi: 10.1073/pnas. No use, distribution or reproduction is permitted which does not comply with
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