Deep-Sea Research Part II 146 (2017) 53–58

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Deep-Sea Research Part II

journal homepage: www.elsevier.com/locate/dsr2

Dominance of Epsilonproteobacteria associated with a whale fall at a T

4204 m depth – South Atlantic Ocean
Angélica Cavaletta, Marcus Adonai Castro da Silvaa, Takashi Toyofukub, Rodrigo Mendesc,
Rodrigo Gouvêa Taketanic, Jéssica Pedrinia, Robert Cardoso de Freitasa,
Paulo Yukio Gomes Sumidad, Toshiro Yamanakae, Yuriko Naganob, Vivian Helena Pellizarid,
⁎
José Angel Alvarez Pereza, Hiroshi Kitazatob, André Oliveira de Souza Limaa,
a
Centro de Ciências Tecnológicas da Terra e do Mar, Universidade do Vale do Itajaí, R. Uruguai 458, 88302-202, Itajaí-SC, Brazil
b
Japan Agency for Marine-Earth Science and Technology - JAMSTEC, 2-15 Natsushima-cho, 237-0061 Yokosuka, Japan
c
Embrapa Meio Ambiente, Rod. SP340 km 127.5, 13820-000 Jaguariúna-SP, Brazil
d
Instituto Oceanográfico, Universidade de São Paulo, Praça do Oceanográfico, 191, 05508-120 São Paulo-SP, Brazil
e
Graduate School of Natural Science and Technology, Okayama University, 700-8330, Okayama, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: The deep ocean is the largest marine environment on Earth and is home to a large reservoir of biodiversity.
Deep sea Within the deep ocean, large organic falls attract a suite of metazoans and microorganisms, which form an
Metagenomics important community that, in part, relies on reduced chemical compounds. Here, we describe a deep-sea
Marine bacteria (4204 m) microbial community associated with sediments collected underneath a whale fall skeleton in the
South Atlantic Ocean. Metagenomic analysis of 1 Gb of Illumina HiSeq. 2000 reads, including taxonomic and
functional genes, was performed by using the MG-RAST pipeline, SEED, COG and the KEGG database. The results
showed that Proteobacteria (79%) was the main phylum represented. The most dominant bacterial class in this
phylum was Epsilonproteobacteria (69%), and Sulfurovum sp. NBC37-1 (97%) was the dominant species.
Different species of Epsilonproteobacteria have been described in marine and terrestrial environments as im-
portant organisms for nutrient cycling. Functional analysis revealed key genes for nitrogen and sulfur cycles,
including protein sequences for Sox system (sulfur oxidation) enzymes. These enzymes were mainly those of the
Epsilonproteobacteria, indicating their importance for nitrogen and sulfur cycles and the balance of nutrients in
this environment.

1. Introduction The microbial communities living in deep-sea sediments are diverse,

despite the extreme conditions prevailing in these habitats. The bac-
Deep-sea environments are the most widespread ecosystems on terial phylum Proteobacteria is one of the most frequently reported in
Earth and are characterized by high hydrostatic pressures, low tem- deep-sea sediments, and all of its classes are represented (Orcutt et al.,
peratures, the absence of sunlight and low nutrient availability 2001). The class Epsilonproteobacteria has been described in marine
(Kennedy et al., 2008; Lalli and Parson, 1997). These ecosystems con- and terrestrial ecosystems as an important component of biochemical
tain different habitats, including hydrothermal vents, cold seeps, tren- and geological processes throughout Earth's history (Campbell et al.,
ches and seamounts, among others (Orcutt et al., 2001). Whale falls are 2006) and includes cultivated organisms such as Sulfurovum, Nitratir-
specialized microbial habitats that occur on the sea floor and are uptor and Sulfurimonas and non-cultivated groups, reported mainly from
formed when a whale carcass sinks to the sediment, introducing large hydrothermal fields (Nakagawa et al., 2005; Mino et al., 2014) but also
amounts of food in an environment that otherwise contains little or- from cold seeps (Pop Ristova et al., 2014).
ganic matter (Smith and Baco, 2003). This creates a sulfur-based che- The deep-sea organisms from Epsilonproteobacteria have the ability
moautotrophic ecosystem (Treude et al., 2009) that supports fauna to grow chemoautotrophically with H2 and/or reduced sulfur com-
including mollusks, crustaceans and polychaetes (Smith and Baco, pounds as electron donors and are involved in biogeochemical cycling
2003). in hydrothermal vent habitats and other sulfur-rich environments.

⁎
Correspondent author.
E-mail address: [email protected] (A.O.d.S. Lima).

http://dx.doi.org/10.1016/j.dsr2.2017.10.012

Available online 21 October 2017

0967-0645/ © 2017 Elsevier Ltd. All rights reserved.
A. Cavalett et al. Deep-Sea Research Part II 146 (2017) 53–58

These microorganisms seems to participate in reduced sulfur oxidation taxonomically and functionally to four published marine metagenomes
by using the so-called Sox System, as reported in studies of the strain using the procedures previously described. Metagenomes were selected
Sulfurovum sp. NBC37-1 (Yamamoto et al., 2010) and other genera according to environmental conditions to investigate the microbial
(Akerman et al., 2013). Microbial degradation of the whale organic community and its relationship to the environment. Selected meta-
matter by sulfate reduction creates sulfide-rich sediments in whale falls genomes included a whale bone metagenome (WF_Bone, MG-
similar to hydrothermal vents and cold seeps (Treude et al., 2009), 4441619.3) - Antarctic Peninsula shelf (Tringe et al., 2005); a microbial
providing appropriate conditions for the development of sulfur-oxi- mat metagenome associated with a whale carcass (WF_Mat, MG-
dizing Epsilonproteobacteria. However, the occurrence of this class in 4441656.4) - Pacific Ocean, Santa Cruz Basin (Tringe et al., 2005); a
whale falls is poorly studied; the class has been found in only one of the whale rib metagenome (WF_Rib, MG-4441620.3) - Pacific Ocean, Santa
three whale falls studied by Tringe et al. (2005) using a metagenomic Cruz Basin (Tringe et al., 2005); a right whale gut microbiome – MG-
approach. 4517288.3 (Sanders et al., 2015); a humpback whale gut microbiome –
In this study, we analyzed environmental DNA from a deep-sea MG-4526723.3 (Sanders et al., 2015); a Brazos-Trinity Basin sediment
(4203 m) sediment sample collected below the vertebral bones of a metagenome (Sediment_1, MG-4441215.3) – Atlantic Ocean, Gulf of
whale fall in the South Atlantic Ocean. The microbial diversity was Mexico (Biddle et al., 2011); an Equatorial Pacific Ocean subseafloor
examined by using the MG-RAST pipeline; and functional key enzymes metagenome (Sediment_2, MG-4516247.3) (D'Hondt et al., 2009); a
were also studied to determine the importance of the microbial com- cellular Hulk hydrothermal metagenome (ThermalVent_1, MG-
munities in this extreme and singular environment. 4481541.3) (Anderson et al., 2014); an Alviniconcha holobiont meta-
genome (ThermalVent_2, MG-4491347.3) (Sanders et al., 2013); an
2. Experimental procedures oxic sediment incubation metagenome (ColdSeep_1, MG-4556390.3) –
cold seep near Barbados; and an ELBA methane seep metagenome
2.1. Sampling, DNA extraction and sequencing (ColdSeep_2, MG-4564112.3).

Sampling occurred during the Iatá-Piúna Cruise, a joint marine re- 2.4. Sulfur measurement
search collaboration between Brazil and Japan, on board the R/V
Yokosuka in April 2013, using the manned submersible Shinkai 6500. For total sulfur (TS) concentration and sulfur isotope measurement,
Marine sediments were collected immediately below the vertebral the pulverized and dried sediment samples were converted into sulfate
bones of a whale fall in the Atlantic Ocean (28°31.1083 S; by heating with hot hydrogen peroxide solution. During the process,
41°39.4081 W), in the deepest part of the São Paulo Plateau (4204 m). iron sulfides (mainly framboidal pyrite) were converted to sulfate ions
Samples were stored at −80 °C until environmental DNA extraction. in solution. The resulting sulfate ions were recovered as BaSO4 by
The total DNA was extracted using the Mo-Bio PowerSoil™ Isolation Kit adding BaCl2 solution, and the TS concentrations were calculated from
(Mo-Bio, CA, USA). DNA samples were pooled together and examined the weight of the recovered BaSO4 precipitate. The BaSO4 sample was
by agarose gel electrophoresis. The sequencing was performed at then mixed with excess V2O5, and the isotopic ratio was measured using
Macrogen (Seoul, Korea) using the Illumina HiSeq. 2000 System a continuous flow-isotope ratio mass spectrometer (IsoPrime™EA, GV
(Paired-End 100 bp), and they received the code Yoko_16. Instruments, UK). Isotope data are reported in the conventional δ34S
notation relative to Vienna Cañon Diablo Troilite. The overall re-
2.2. Taxonomic and functional key enzyme analysis producibility of the sulfur isotopic analysis was ± 0.2‰.

Metadata were uploaded (4558538.3) to the MG-RAST pipeline 3. Results and discussion
(Meyer et al., 2008; Smedile et al., 2013; Tang et al., 2013) and pro-
cessed according: filter sequences based on length, number of ambig- 3.1. Microbial diversity in Yoko_16
uous bases and quality values; removal of sequencing artifacts and DNA
contamination; RNA identification and analysis; identification of pu- To analyze the DNA composition of the microbial community from
tative protein coding feature, aminoacid clustering and protein anno- Yoko_16, the extracted DNA was sequenced, and the reads were up-
tation. Protein sequences were annotated using the MG-RAST pipeline loaded to MG-RAST. The Illumina sequencing produced 10.4 million
(Best Hit Classification and Hierarchical Classification tools). For reads (Phred > Q30 = 96.54%) with a GC content of 38%. The dataset
taxonomic analysis, datasets were compared against the SEED database, uploaded to MG-RAST contains 1,709,787 sequences with an average
using an E-value cutoff of 10−5, an identity cutoff 60%, an alignment length of 158 bp. Of the sequences tested, 44,307 sequences (2.59%)
length of 15 bp and raw values. The results were exported in table failed to pass the MG-RAST´s Quality Control (QC) pipeline. Of those,
format and analyzed individually. The metagenomic functional ana- dereplication identified 34,645 sequences as artificial duplicate reads.
lyses were performed using the Hierarchical Classification tool, against Of the sequences that passed QC, the platform processed 78% of the
the COG database, with a max. E-value cutoff of 10−5, a min. identity sequences as annotated proteins; 3% were ribosomal genes, and 13%
cutoff of 60% and a min. alignment length cutoff of 15 bp. A table was were proteins of unknown function. The number of proteins sequences
made listing key functional enzymes described in other marine meta- with unknown function were higher in YK_16 than what was observed
genomics studies (Quaiser et al., 2011; Smedile et al., 2013). These in WF_Bone (0%), WF_Mat (0.2%) and WF_Rib (3.7%), probably due to
enzymes were used as queries to search each metagenome, and the MG- the larger number of sequences analyzed. In fact, the advances in DNA
RAST abundance was recorded. We also searched specifically for en- sequencing technologies propitiated a huge increase in number of
zymes relevant to nitrogen and sulfur cycling using KEGG (MG-RAST genomes and protein sequences available in public databases. For in-
pipeline). The sequences found in the metagenomes were recognized stance, in July 2015, the UniProtKB / TrEMBL database had more than
manually using the COG database. These sequences were transferred to 50 million sequences (European Bioinfomatics Institute Website http://
the SEED database to determine which microorganisms have genes that www.ebi.ac.uk/). The number of possible protein sequences is astro-
encode the protein sequence. The rarefaction curve was created by nomically large, it is being estimated to be around 1012 sequences
using the MG_RAST pipeline (data not shown). among prokaryotes (Godzik, 2011), and most of them we still do not
know their function. Currently known sequences are analyzed in terms
2.3. Diversity and functional comparison with other marine metagenomes of its primary sequence, families that have single-domain or multi-
domain architectures and whether they have a known three-dimen-
The metagenome from Yoko_16 (MG-4558538.3) was compared sional structure (Levitt, 2009). However, just a fraction of the sequence

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A. Cavalett et al. Deep-Sea Research Part II 146 (2017) 53–58

possibility are already characterized molecularly. Thus, we still have a Table 1

long way to cover all possibilities, and the discovery of the functions of Data processed by MG-RAST platform relative to the metagenomes selected: WF_Bone:
whale bone metagenome; WF_Mat: microbial mat metagenome associated to whale car-
these unknown proteins is one of the great challenges of the post-
cass; and WF_Rib: whale rib metagenome.
genomic biology.
The SEED database results showed that the most abundant organ- Yoko_16 WF_Bone WF_Mat WF_Rib
isms were Bacteria, and most sequences were from Proteobacteria
Sequence reads (n) 1 709 787 40 549 38 281 38 496
(76%). Unlike other marine metagenomes, Epsilonproteobacteria
Average length (pb) 158 1017 990 1016
(69%) were predominant among the Proteobacteria. Quaiser et al. Processed data (%) 97.4 100 10 100
(2011) described Planctomycetes, Delta and Gammaproteobacteria Known proteins (%) 78.1 100 87.5 95.5
(Proteobacteria) as the main groups present in marine sediments from Ribosomal genes (%) 3.1 0 0.4 0.8
the Sea of Marmara using the small subunit ribosomal RNA (SSU rRNA). Unknown proteins (%) 13.5 0 0.2 3.7
Unknown sequences (%) 2.7 0 0 0
In addition, Smedile et al. (2013) identified the Proteobacteria (Gam-
Sequencing method Illumina Sangera Sangera Sangera
maproteobacteria) as the most abundant microorganisms in the deep Number of species (n) 3909 2098 2168 2211
Mediterranean Sea (4908 m depth). The high abundance of DNA se- Data analyzed (Gb) 0.27 0.04 0.04 0.04
quences from Epsilonproteobacteria in Yoko_16 is probably related to
a
the environment, as the same bacteria were found to be abundant in Sequenced by standard protocols (http://www.jgi.doe.gov/) (TRINGE et al., 2005).

deep marine environments and in transition zones of oxygenated and

anoxic marine environments (Campbell et al., 2006). In the present both the WF_Bone and WF_Rib metagenomes, the most abundant class
study we were unable to do a detailed chemical characterization of the was Gammaproteobacteria. Both of these metagenomes come from
samples studied but, as reported by Treude et al. (2009) on the Cali- whale bones from different geographic locations than Yoko_16. For
fornia Margin, sediments near a whale fall had high microbial activities, WF_Mat, Alphaproteobacteria was the most prevalent group. All these
high sulfate reduction rates, and abrupt chemical gradients, which may studies were carried out in different ocean areas from Yoko_16, and
favor the occurrence of Epsilonproteobacteria. Besides, Epsilonproteo- none of the metagenomes were from marine sediments underneath a
bacteria has also been described in anaerobic and microaerophilic, substrate. The Yoko_16 microenvironment may be anaerobic or mi-
sulfur-rich marine environments, in symbiosis with metazoans, deep- croaerophilic, which may be why there was a high number of sequences
sea hydrothermal vent fluids, deep-sea caves rich in sulfur, and other from Epsilonproteobacteria, a class that has an important role in nu-
sites (Meyer et al., 2013; Meyer and Huber, 2014; Rossmassler et al., trient cycling in such locations. Epsilonproteobacteria has been de-
2012). All of these works support our report on the dominance of Ep- scribed in other marine habitats such as deep-sea hydrothermal vent
silonproteobacteria in this South Atlantic whale fall. fluids and vent-associated subsurfaces (Nakagawa et al., 2005, 2006;
At a lower taxonomic level, most of the sequences (97%) from Takai et al., 2004), suggesting the importance of this class in extreme
Epsilonproteobacteria were related to Sulfurovum sp. NBC37-1. BLAST environments.
analysis from Sulfurovum sp. NBC37-1 ribosomal RNA sequences (1539) The microbial groups detected in Yoko_16 include organisms from
found in the present work revealed 88–99% identity with Sulfurovum the sediments, but they may also be derived from the microbiota of the
sp. NBC37-1 ribosomal RNA sequences deposited by Nakagawa et al. whale itself. Sanders et al. (2015) studied the microbiome of a baleen
(2007). The wide range of similarity suggests that the DNA sequences whale and reported the dominance of the phylum Firmicutes among
assigned to Sulfurovum sp. NBC37-1 are not exclusively from organisms others, and Proteobacteria was a minor member of the microbiota in
of this species; in some cases, even the genus may be different. The that animal. To further elucidate this relationship, the Yoko_16 meta-
whole-genome sequence of Sulfurovum sp. NBC37-1 (accession number genome was compared to the metagenomes derived from whales,
AP009179) collected from deep-sea vents was determined by marine sediments, cold seeps, thermal vents and other whale falls by
Nakagawa et al. (2007), and key genes associated with sulfur reduction principal components analysis (Fig. 2). Results from whale fall meta-
and oxidation were identified. Yamamoto et al. (2010) reported that the genomes analysis suggests to be more similar to cold seeps and thermal
enzyme for sulfur oxidation was constitutively expressed. However, the vents than to the whale metagenomes. Moreover, there was a large
enzymes for sulfur reduction were expressed depending on physical and difference between Yoko_16 and the whale metagenomes in terms of
chemical conditions. This strain is capable of growth via hydrogen and their main phyla, Proteobacteria and Firmicutes. This may indicates
sulfur-compound oxidation under microaerobic and anaerobic condi- that the microorganisms living in whale fall communities are probably
tions; it has no dissimilatory sulfite reductase, adenylylsulfate reductase not derived from the whale itself. Rather, they are probably enriched by
or adenylylsulfate: phosphate adenyltransferase (Nakagawa et al., the presence of the carcass and reinforce the association of the com-
2007). munity with sulfur-rich ecosystems.

3.2. Microbial Diversity Comparison of Yoko16 to other marine 3.3. Nitrogen and sulfur metabolism
metagenomic data
We searched for key sequences associated with nitrogen and sulfur
In this study, we compared four published marine metagenomes to metabolism in the dominant Epsilonproteobacteria found in the present
the Yoko_16 data using the MG-RAST platform and the SEED database. study, because this class has been described as important in the cycles
Metadata from WF_Bone, WF_Mat, WF_Rib, and Sanger sequencing of these elements (Vetriani et al., 2014; Yamamoto et al., 2010). Ni-
showed a comparable number of sequences and similar average length, trogen and sulfur metabolism in the Yoko_16 data were evaluated using
and the processed data and percentage of annotated proteins in the data the KEGG tool from the MG_RAST platform and the SEED database.
obtained were similar to the results obtained from Illumina sequencing The metagenome Yoko_16 contains key enzyme sequences for both
(Yoko_16) (Table 1). The results were similar because of the sequencing the nitrogen and sulfur cycles. Regarding the former, there are different
method used by the researchers, which produced different volumes of sequences for nitrate reductase (NADH) and nitrite reductases (NO-
data to analyze, and different numbers and sizes of sequences were forming, NAD(P)H, Ferredoxin, cytochrome; ammonia-forming). These
generated. sequences are from Euryarchaeota, Bacteroidetes, Firmicutes,
All the metagenomes have Proteobacteria as the most prevalent Proteobacteria and organisms not yet assigned to any described phyla.
phylum: WF_Bone 66,08%, WF_Mat 79,84%, and WF_Rib 83,03%. The WF_Bone metagenome did not contain sequences for nitrate re-
Metagenome evaluation showed that Epsilonproteobacteria is the most ductases, and the nitrite reductase sequences were exclusively related
abundant class in Yoko_16, unlike the other metagenomes (Fig. 1). In to phylum Proteobacteria. For WF_Mat, the nitrate and nitrite

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A. Cavalett et al. Deep-Sea Research Part II 146 (2017) 53–58

Fig. 1. occurrence of different Proteobacterial taxa based on the

taxonomic binning of protein-encoding genes against the SEED da-
tabase using MG-RAST pipeline. The taxonomic assignment SEED
database was carried out using an E-value cutoff 10−5, Identity
cutoff 60% and Alignment Lengh of 15pb. WF_Bone: bone whale
carcass metagenome (MG-4441619.3); WF_Mat: microbial mat me-
tagenome associated to whale carcass (MG-4441656.4); WF_Rib: rib
bone whale carcass metagenome (MG-4441620.3) and Yoko_16
(MG-4558538.3).

sequences primarily belonged to phylum Proteobacteria, and the in the Yoko_16 environment, by using nitrate as an electron acceptor in
WF_Rib metagenome had sequences for nitrate reductase classified low oxygen or anoxic conditions
mostly as Alphaproteobacteria, and the largest portion of sequences for For the sulfur cycle, we observed enzymes involved in both sulfur
nitrite reductase were unassigned. The difference in dominant phyla reduction and oxidation: sulfate adenylyltransferase, sulfite oxidase,
may be related to the nature of the sample and the environmental adenylyl-sulfate reductase, sulfite reductase (NADPH and ferredoxin)
conditions, and, most importantly, the low oxygen concentration in the and adenylyl-sulfate kinase. Sulfurovum sp. NBC37-1
sediment below the whalebone. In the Yoko_16 metagenome, the ni- (Epsilonproteobacteria) is the main species that contains the cited
trate reductase sequences were related to Arcobacter butzleri and genes, which corroborates the importance of Epsilonproteobacteria for
Sulfurimonas denitrificans, both of which belong to nutrient cycling in this environment. Campbell et al. (2006) described
Epsilonproteobacteria. The nitrite reductase sequences were mostly Epsilonproteobacteria as an important group in modern vents and si-
related to Desulfotalea psychrophila (Deltaproteobacteria) and milar extreme habitats, stating that it plays a significant role in geolo-
Sulfurovum sp. NBC37-1 (Epsilonproteobacteria). Furthermore, 16% of gical and biogeochemical processes. Enzymes related to sulfur reduc-
the annotated sequences for nitrite reductase were classified as unas- tion could be used by these organisms to obtain energy from donors as
signed. Some studies reported that the genes for nitrate reductase hydrogen in the absence of oxygen or nitrate as electron acceptors, as
subunits (α-, β- and λ-) and the nar system for nitrate respiration are reported for Sulfurimonas paralvinellae (Takai et al., 2006) and other
depth-dependent (Papaspyrou et al., 2014). The presence of these genes similar bacteria (Nakagawa et al., 2005). It has been proposed that the
also suggests that the deep-sea microbial communities are better Sox system or thiosulfate oxidation system, which were studied in the
adapted under microaerophilic environments in possible association alphaproteobacterium Paracoccus pantotropus (Friedrich et al., 2005,
with particulate organic matter (POM) from the epipelagic zone 2007, 2001), require at least four soluble proteins, SoxAX, SoxYZ, SoxB
(Arístegui et al., 2009; Eloe et al., 2011; Ivars-Martinez et al., 2008; and Sox(CD)2, for the total oxidation of thiosulfate (Friedrich et al.,
Konstantinidis et al., 2009). In extreme environments, Pérez-Rodríguez 2001). According to Nakagawa et al. (2007) and Sievert et al. (2008),
et al. (2013), observed nitrate reductase sequences from the genus Epsilonproteobacteria strains (NBC37, SB155-2 and DSM 125) from
Marinobacter, in the Gammaproteobacteria, as the most prevalent in sulfide- and sulfur-rich environments also have sox gene clusters. Gene
libraries of clones from a black smoker and a diffuse flow vent. The sequences from the sox system (soxA, soxB, soxD, soxH, soxX, soxY,
nitrate and nitrite reductases sequences observed in Yoko_16 show the soxW e soxZ) were found in Yoko_16 (total sulfur of 0.32% and δ34 S of
importance of Epsilonproteobacteria for part of the nitrogen cycling at 4.1‰), mostly related to Sulfurimonas denitrificans from

Fig. 2. A-Principal component analysis ordination of individual

metagenomes based taxonomic affiliations of reads at phylum
level. B-Mean and error bars comparing the composition of
Proteobacteria and Firmicutes in whale carcass metagenomes and
whale gut microbiomes. The metagenomes were arranged into
categories as follows: Whale carcass: Yoko_16 (MG-4558538.3),
WF_Bone (MG-4441619.3), WF_Mat (MG-4441656.4), WF_Rib
(MG-4441620.3); Whale microbiome: Right_Whale (MG-
4517288.3), Humpback_Whale (MG-4526723.3); Thermal vent:
ThermalVent_1 (MG-4481541.3), ThermalVent_2 (MG-
4491347.3); Cold seep: ColdSeep_1 (MG-4556390.3), ColdSeep_2
(MG-4564112.3); Sediment: Sediment_1 (MG-4441215.3),
Sediment_2 (MG-4516247.3).

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A. Cavalett et al. Deep-Sea Research Part II 146 (2017) 53–58

Table 2 Elifantz et al., 2005). Enzymes involved in the degradation of biopo-

Comparison of gene abundances for key metabolic enzymes in Yoko_16 metagenome with lymers (alpha/beta glucosidases, leucyl-aminopeptidases and ar-
those from: WF_Bone: whale bone metagenome; WF_Mat: microbial mat metagenome
ylsulfatases) and in phosphorus uptake (ABC-type phosphate transport
associated to whale carcass; and WF_Rib: whale rib metagenome. The functional genes
assignment was carried out using COG database, E-value cutoff 10−5, Identity cutoff 60% systems) were found in the Yoko_16 metagenome. The sequences for all
and Alignment Length of 15pb. these proteins were more abundant in Yoko_16 (~37% of the proteins
sequences evaluated) than in other reported whale carcass metagen-
Functional Enzymesa Yoko_16 WF_Bone WF_Mat WF_Rib omes (~14% in the WF_Bone metagenome).
Ammonia permease 1027 20 20 43 Enzymes responsible for anaplerotic activities via restocking of ox-
Leucyl aminopeptidase 445 33 20 23 aloacetate to the rTCA/TCA cycle, phosphoenolpyruvate carboxylase
Lipase 291 31 24 19 and phosphoenolpyruvate carboxykinase (ATP) (Nelson et al., 2008)
ABC-type phosphate transport 335 23 8 25 were also found in large numbers (8% of the evaluated protein se-
Alpha-glucosidasesb 106 8 2 0
quences) in the Yoko_16 metagenome. We detected genes for carbon
Beta-glucosidase-related glycosidases 557 39 22 22
β-glucosidasec 13 1 6 1 monoxide dehydrogenase in all of the metagenomes that were ana-
Arylsulfatase A and related enzymes 3717 38 42 27 lyzed. The oxidation of CO to CO2 has been studied in the litotrophy as
Carbon monoxide dehydrogenased 174 40 30 19 an alternative or supplementary energy source for heterotrophy
Chaperonin GroEL (HSP60 family) 788 23 22 21
(Martin-Cuadrado et al., 2009). Many marine bacteria have been de-
Phosphoenolpyruvate carboxylase 131 26 8 12
Phosphoenolpyruvate carboxykinasee 915 22 8 17 scribed in this situation, including members of the Roseobacter clade
Pyruvate carboxyltransferase 716 7 4 11 (Brinkhoff et al., 2008; King and Weber, 2007).
Transposases 3784 509 153 370
4. Concluding remarks
a
MG_RAST Abundance < .
b
Glycosyl hydrolases family 31.
c
6-phospho-β-glucosidase/β-galactosidase.
We identified Epsilonproteobacteria as the main class from the do-
d
Aerobic-type, CoxL/CutL. main Bacteria in the Yoko_16 environment. The prevalence of this class
e
ATP. seems to be connected to the environmental conditions, i.e., deep-sea
sediments below a whalebone and the nutrient cycling that occurs
Epsilonproteobacteria. We detected a sox cluster with the genes soxX, there. DNA sequences from the microorganisms in this environment
soxY, soxZ, soxA and soxB through assembly analysis performed by CLC show the ability of these organisms to assimilate, reduce, and to oxidize
software. Compared to other marine metagenomes, Yoko_16 was the sulfur and nitrogen. The ability of Epsilonproteobacteria to produce
only one with sequences for the Sox System (sulfur oxidation). This energy from inorganic compounds is the reason for their dominance in
could be explained by the elevated number of sequences from Epsi- a dark, cold and organic-poor environment.
lonproteobacteria, in which species with genes for the Sox System have
been described before (Sievert et al., 2008; Yamamoto et al., 2010). Acknowledgments

CAPES (Brazil, Process CAPES/JSPS 02/13), FAPESC (Brazil,

3.4. Key functional enzymes from the Yoko_16 metagenome Process 3422/2012) and CNPq - INCT-Mar COI (Brazil, Process
565062/2010-7) supported this work. We also thank CAPES for scho-
We searched for enzymes involved in cellular metabolism and in- larship support provided to A.C. (PROSUP Program) and A.O.S.L
formation storage and processing to determine the energy sources and (Process 08740/14-3); CNPq for scholarship provided to A.O.S.L
adaptation skills used by the microorganisms in the Yoko_16 meta- (Process 311010/2015-6).
genome. We identified a large number of transposase sequences
(29.1%) compared to protein sequences in the functional gene analysis Compliance with ethical standards
for the Yoko_16 metagenome (Table 2). The same was observed for
other deep-sea microorganism metagenomes (Qin et al., 2011; Smedile The authors declare that they have no conflict of interest. This ar-
et al., 2013). The high number of transposable elements may be due to ticle does not contain any studies with human participants or animals
the important role of transposases in allowing hosts to face environ- performed by any of the authors.
mental challenges (Casacuberta and González, 2013). Among the dif-
ferent mechanisms by which transposases support the host genomes, we References
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