Genome sequence of a native-feather degrading extremely thermophilic Eubacterium, Fervidobacterium islandicum AW-1
Lee et al. Standards in Genomic Sciences
Genome sequence of a native-feather degrading extremely thermophilic Eubacterium, Fervidobacterium islandicum AW-1
Yong-Jik Lee 0
Gun-Seok Park 0
Yunyoung Kwak 0
Sang Jun Lee
Min-Kyu Park 0
Ji-Yeon Kim 0
Hwan Ku Kang
Jae-Ho Shin 0
Dong-Woo Lee 0
0 School of Applied Biosciences, Kyungpook National University , Daegu , Korea
Fervidobacterium islandicum AW-1 (KCTC 4680) is an extremely thermophilic anaerobe isolated from a hot spring in Indonesia. This bacterium could degrade native chicken feathers completely at 70 °C within 48 h, which is of potential importance on the basis of relevant environmental and agricultural issues in bioremediation and development of eco-friendly bioprocesses for the treatment of native feathers. However, its genomic and phylogenetic analysis remains unclear. Here, we report the high-quality draft genome sequence of an extremely thermophilic anaerobe, F. islandicum AW-1. The genome consists of 2,359,755 bp, which encodes 2,184 protein-coding genes and 64 RNA-encoding genes. This may reveal insights into anaerobic metabolism for keratin degradation and also provide a biological option for poultry waste treatments.
Native feather; Keratin; Degradation; Extremophile; Fervidobacterium islandicum AW-1
Keratin, a key structural material in feathers, skin, hair,
nails, horns, and scales, is one of the most abundant
proteins on earth, and it is a mechanically durable and
chemically unreactive protein. Since feather keratin
contains a high content of cysteine (~7 %) in its amino acid
sequence, it has a strong and fibrous matrix through
disulfide bonds. Such a highly rigid, strongly cross-linked,
indigestible polypeptide has very limited industrial
applications due to its rigidity and indigestibility, and is thus
often considered a solid waste. In fact, more than 5
millions of tons of chicken feathers in poultry industry are
generated globally every year, and such waste by-products
can cause a serious solid waste problem [1, 2]. At present,
most waste chicken feathers are disposed by burning,
burying in landfills or recycling into low quality animal
feed. However, these disposal methods are restricted due
to increase in greenhouse gas emissions and
environmental pollution. Many efforts aimed at meeting
environmental performance criteria and renewable energy production
are in progress to degrade poultry feathers to soluble
peptides and amino acids for the use of fertilizers,
animal feedstock, and soil conditioner . Thus,
development of a bioconversion process for degradation
of feathers will provide considerable opportunities for
industrial applications [4, 5]. In this regard,
keratinolytic microorganisms have great importance in feather
waste degradation and its use for improvement of
livestock feed and production of hydrolysates. Hence,
many microbial keratinases, differing from commonly
known proteases (e.g., trypsin, pepsin and papain),
have been sought to hydrolyze this recalcitrant
polypeptide. Toward this aim, several keratin-degrading
microorganisms, including Bacillus licheniformis PWD-1 ,
Aspergillus fumigatus , and Streptomyces pactum DSM
40530  have been isolated and characterized.
Nevertheless, the efficiency and feasibility of such bioprocesses is
still limited in terms of practical applications, mainly due
to the instability of enzyme activity, low yields of keratin
degradation, and its long process time.
Previously, we isolated an extremely thermophilic
bacterium from a geothermal hot spring in Indonesia .
When grown in TF medium supplemented with 0.8 %
(w/v) of native chicken feathers, this bacterium could
degrade native chicken feathers completely within 48 h
© 2015 Lee et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
at 70 °C under anaerobic conditions. Morphological,
physiological and 16S rRNA gene sequencing analyses
demonstrated that this native chicken feather degrading
bacterium belonging to the genus Fervidobacterium was
identified as Fervidobacterium islandicum AW-1 .
Moreover, it was found that adding the reducing reagent
greatly hastened the degradation of native chicken
feathers, indicating that breakage of disulfide bonds are
also responsible for the complete degradation of feather
keratin. Therefore, we hypothesized that not only
keratinolytic proteases but also other enzymes specific to
disulfide bonds might be mainly involved in degradation
of keratin. Accordingly, these and related reasons led us
to sequence the whole genome of F. islandicum AW-1,
providing an insight into the degradation of
nondigestible keratin biomass. Moreover, comparative
genomics for feather-degrading F. islandicum AW-1 and its
closely related non-degrading bacteria will shed light on
the evolutionary relationship between them. Here, we
present a summary of classification and a set of general
features for F. islandicum AW-1 together with the
description of genome properties and annotation.
Classification and features
Out of 37 native chicken feather-degrading anaerobic
strains grown at 70 °C enriched in EM-1 medium
supplemented with native chicken feathers as a
carbon source, we chose the strain AW-1 showing the
highest keratinolytic activity . Subsequently, we identified
the strictly anaerobic, rod shaped (0.6 × 1 ~ 3.5 μm), motile,
non-sporulating, Gram-negative extremophilic bacterium
as Fervidobacterium islandicum AW-1 based on cell
morphology, physiological characteristics, common DNA
characteristics, 16S rRNA gene sequence, and cellular fatty
acid profile as described previously (Fig. 1a, b) . This
bacterium belongs to the order of Thermotogales, of
which all members are Gram-negative rod-shaped
anaerobic extremophiles containing unique lipids .
After the first isolate F. nodosum had been reported, several
Fervidobacterium strains including F. islandicum , F.
gondwanense , F. pennivorans , F. changbaicum ,
and F. riparium  were isolated and characterized. All of
them grew on glucose, mainly producing H2, CO2, and
acetate, and also fermented a wide range of nutrients such as
peptone, yeast extract, pyruvate, glucose, maltose,
raffinose, and starch. Such organotrophs can also reduce S0
to H2S during the course of fermentation. In
particular, F. islandicum AW-1 showed the highest
keratinolytic activity, resulting in the complete degradation of
native chicken feathers (8 g/L) within 48 h (Fig. 1b),
and its optimal growth temperature and pH on the
native feathers were 70 °C and pH 7.0, respectively
. Among the genus Fervidobacterium, F. islandicum
AW-1 together with F. pennivorans have been found
as native-feather degrading bacteria [9, 13]. Fig. 2
shows the phylogenetic neighborhood of F. islandicum
AW-1 in a 16S rRNA gene sequence-based tree. This
strain clusters closest to the genus of Fervidobacterium,
the Thermotogales order. The 16S rRNA gene sequence
(1456 bp) of F. islandicum AW-1 obtained from its
genome sequence showed high levels of sequence similarity
with members of the genus Fervidobacterium, such as
F. changbaicum (99.3 %) , F. pennivorans (98.1 %)
, F. islandicum (97.3 %) , F. riparium (96.1 %)
, F. gondwanense (94.7 %)  and F. nodosum
(95.4 %)  (Fig. 2). RAST analysis to rapidly call
and annotate the genes of a complete or essentially
complete prokaryotic genome  also suggested that
F. nodosum Rt17-B1 was actually F. islandicum AW-1's
Fig. 1 a The scanning electron micrographs (SEM) of F. islandicum AW-1 grown on the TF medium supplemented with glucose (0.5 %, w/v)
during anaerobic fermentation at 70 °C. b Complete degradation of native feathers by F. islandicum AW-1. The cells were grown on the TF
medium supplemented with native feathers (0.8 %. w/v) during anaerobic fermentation at 70 °C for 48 h. For the preparation of specimens
for F. islandicum AW-1, we followed the protocol as described previously
Fig. 2 Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of F. islandicum AW-1 (in bold) to members of the family
Thermotogaceae. The evolutionary history was inferred using the Neighbor-Joining method. The analysis involved 36 nucleotide sequences.
All positions containing gaps and missing data were eliminated. There were a total of 1,235 positions in the final dataset. Bootstrap values
(percentages of 1,000 replications) are shown next to the branches. The sequences used in the analysis were obtained from the GenBank
database. Bar, 2 nt substitution per 100 nt. Evolutionary analyses were conducted in MEGA6
closest neighbor. ANI analysis using BLAST  showed
that, among the completely sequenced Fervidobacterium
and Thermotoga species, F. pennivorans was closest to
F. islandicum AW-1 (77.4 % sequence identity and 78.9 %
alignment). As shown in Fig. 1, this strain was rod-shaped,
occurring singly, in pairs or short chains with a single
polar spheroid, a sheath-like outer membrane structure, a
so called “toga”, which is a typical morphological
feature belonging to the order of Thermotogales.
Together with the previous phenotypic and biochemical
next-generation sequencing was performed at Pacific
Biosciences (Menlo Park, CA). The assembly and
annotation were performed by using the hierarchical
genomeassembly process  protocol RS HGAP Assembly 2 in
SMRT analysis version 2.2.0 (Pacific Biosciences), NCBI
COG  and RAST server database . The whole
complete genome sequence of F. islandicum AW-1 has
been deposited at DDBJ/EMBL/GenBank under the
accession number. The AW-1 strain is also available from the
Korean Collection for Type Cultures (KCTC, Daejeon,
Korea). A summary of the project information is shown
in Table 2.
Growth conditions and genomic DNA preparation
F. islandicum AW-1 was grown in TF medium which
contained the following: 0.5 % glucose (instead of 0.8 %
native chicken feather), 1 g of yeast extract, 1.6 g of
K2HPO4, 0.8 g of NaH2PO4 · H2O, 0.16 g of MgSO4 ·
7H2O, 0.1 g of NH4Cl, 1 % (v/v) vitamin solution (2 g of
biotin, 2 g of folic acid, 10 g of pyridoxine-HCl, 5 g of
thiamine-HCl, 5 g of riboflavin, 5 g of nicotinic acid, 5 g
of calcium pantothenate, 0.1 g of vitamin B12, 5 g of
paminobenzoic acid, 5 g of lipoic acid per liter), 1 % (v/v)
trace element solution (2 g of nitrilotriacetic acid, 0.18 g
of ZnSO4 · 7H2O, 3 g of MgSO4 · 7H2O, 0.5 g of MnSO4 ·
2H2O, 1 g of NaCl, 0.1 g of FeSO4 · 7H2O, 0.01 g of
H3BO3, 0.18 g of CoSO4 · 7H2O, 0.01 g of CuSO4 · 5H2O,
0.1 g of CaCl2 · 2H2O, 0.1 g of AlK(SO4)2 · 12H2O,
0.001 g of Na2SeO3 · 5H2O, 0.025 g of NiCl2 · 6H2O,
0.01 g of Na2MoO4 · 2H2O per liter), 1 mg of resazurin
and 0.75 g of Na2S · 9H2O per liter at pH 7 and 70 °C.
The media were prepared as follows; under the N2 gas
Table 2 Project information
MIGS 31.2 Fold coverage
Source material identifier KCTC 4680
10 kb SMRT library
RS HGAP assembly protocol in
SMRT analysis pipeline v.2.2.0
NCBI prokaryotic genome annotation
characterization , our sequence analysis suggested
that this AW-1 strain could be assigned as a native
feathers degradable strain of F. islandicum. This was
also supported by the previous DNA-DNA hybridization
analysis with F. islandicum (92.4 %)  and F. pennivorans
(42 %) .
Genome sequencing information
Genome project history
This bacterium was selected for sequencing to unveil the
degradation mechanism of keratin through transcriptomic
analysis and comparative genomics based on its ability to
completely decompose native feathers under anaerobic
conditions at elevated temperatures (Table 1, Fig. 1b). The
Table 1 Classification and general features of Fervidobacterium
islandicum AW-1 
MIGS ID Property
Family Fervidobacteriaceae TAS 
(Type) strain: AW-1
Optimum temperature 70 °C
pH range; Optimum
Geothermal hot stream
MIGS-22 Oxygen requirement Anaerobic
MIGS-15 Biotic relationship
aEvidence codes - IDA Inferred from Direct Assay, TAS Traceable Author
Statement (i.e., a direct report exists in the literature), NAS Non-traceable
Author Statement (i.e., not directly observed for the living, isolated sample,
but based on a generally accepted property for the species, or anecdotal
evidence). These evidence codes are from the Gene Ontology project 
Genbank date of release December 04, 2014
flushing, adjusted to 7 with 2 N HCl (NaOH), and
sterilized by autoclaving at 121 °C for 20 min prior to use .
The genomic DNA was isolated from a 12 h-grown cells
(5 ~ 7 × 108 cells/ml) in TF medium (0.5 L) using a
QIAmp DNA mini kit (QIAGEN).
Genome sequencing and assembly
Genome sequencing was performed using a single
molecule real-time sequencing platform on PacBio RS II
instrument with P4-C2 chemistry (Pacific Biosciences,
Menlo Park, CA) . Preprocessing of reads and de novo
assembly were performed using the hierarchical
genomeassembly process  protocol RS HGAP Assembly 2 in
SMRT analysis version 2.2.0 (Pacific Biosciences).
Standard parameters were applied as follows: PreAssembler v2
(Minimum Seed Read Length : 6,000 bp) was conducted
then Celera Assembler v1 (Genome Size : 2,500,000 bp,
Target Coverage : 30, Overlapper Error Rate : 0.06,
Overlapper Min Length : 40, Overlapper K-mer : 14) was
performed . We assembled 169,795 reads
(achieving ~351.41 fold coverage) into 12 contigs over 2,000 bp.
The total contig length, maximum contig size, average
contig length, and N50 were 2,359,755 bp, 2,232,638 bp,
196,624 bp, and 2,232,638 bp, respectively (40.74 % G + C)
(Fig. 3 and Table 3).
The genes in the assembled genome were annotated using
NCBI COG . Additionally, automatic functional
annotation of genes was conducted using the RAST server
database . Genes were predicted using GeneMarkS
 as a part of the NCBI prokaryotic genome automatic
Fig. 3 Graphical linear map of the genome of F. islandicum AW-1 strain. From the bottom to the top of each scaffold: Genes on the forward
strand (color by COG categories as denoted by the IMG platform), Genes on the reverse strand (color by COG categories), RNA genes (tRNAs
green, sRNAs red, other RNAs black), GC content, GC skew
Table 4 Number of genes associated with general COG
Table 3 Genome statistics
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
annotation pipeline (PGAAP) . Besides functional
annotation for protein coding genes, PGAAP also
provided information for RNA genes and pseudo genes.
BLASTCLUST parameters for identifying internal clusters
were ‘-L .8 –b T –S 50’. Proteins with Pfam domains, signal
peptides, and transmembrane helices were identified using
InProScan search against HMMPfam , SignalPHMM
, TMHMM  via Blast2Go service . Additional
gene prediction and functional annotation were carried
out using Integrated Microbial Genomes (IMG-ER)
The total size of the genome is 2,359,755 bp, slightly
larger than those of other sequenced Fervidobacterium
strains and G + C content is 40.7 % (Table 3). A total of
2,184 protein coding genes were predicted in 2,248 total
numbers of genes, indicating that 64 RNAs sequences
were identified and 361 of protein coding genes were
assigned to a putative function with the remaining
annotated as hypothetical proteins. The detailed properties
and the statistics of the genome as well as the
distribution of genes into COG functional categories are
summarized in Tables 3 and 4.
Insights from the genome sequence
As described above, the 16S rRNA gene sequence of F.
islandicum AW-1 showed the high similarity to those of
F. changbaicum CBS-1, and F. islandicum H-21. On
the other hand, RAST analysis demonstrated that F.
nodosum Rt17-B1 was actually F. islandicum AW-1's
closest neighbor. Consequently, genome analysis found
genes involved in protein metabolism including protein
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport
General function prediction only
The total is based on the total number of protein coding genes in the genome
degradation systems with 25 different types of proteases. For
example, protein-coding genes annotated as
carboxylterminal protease (EC 184.108.40.206) and lipoprotein signal
peptidase (EC 220.127.116.11) were found in F. islandicum AW-1, but
not in F. nodosum Rt17-B1. We also found several genes
encoding cysteine desulfurase and thioredoxin-disulfide
reductase as potential candidates for feather degradation. In
addition, several reductases and peptidases (e.g., disulfide
reductase, thioredoxin, and carboxy-peptidases) of F.
islandicum AW-1 showed relatively low levels of sequence
identity (less than 50 %) to those of F. nodosum Rt17-B1. In
addition, F. islandicum AW-1 seems to have several distinct
enzymes involved in amino-sugars (chitin and
N-acetylglucosamine) utilization and sugar alcohols (glycerol and
glycerol-3-phosphate) metabolism, which are not found in
F. nodosum Rt17-B1 (Fig. 4). Notably, comparative analysis
of the F. islandicum AW-1 and F. nodosum RT17-B1
genomes revealed that the former seems to have several
distinct enzymes involved in fatty acid degradation, aromatic
Fig. 4 Overview of the microbial pathways on the KEGG pathways using the iPath. Metabolic pathways found in the context of F. islandicum AW-1
(top panel) and F. nodosum Rt17-B1 (bottom panel) genomes are shown in red and blue, respectively. Hypothetical proteins found are excluded
compound degradation, and alpha-linolenic acid
metabolism not found in the latter.
Previously, it was found that addition of the reducing
reagent greatly hastened the degradation of native feathers,
indicating that breakage of disulfide bonds are also
responsible for the complete degradation of feather keratin,
implying that not only keratinolytic proteases but also
other enzymes specific to disulfide bonds might be mainly
involved in degradation of keratin . Indeed, comparison
of the genome sequence of F. islandicum AW-1 with that
of F. nodosum Rt17-B1 suggests that several candidate
enzymes including cysteine desulfurase and
thioredoxindisulfide reductase may be involved in native feather
degradation. In addition, the genome of F. islandicum AW-1
reveals that this strain also possesses some hydrogenases.
Therefore, F. islandicum AW-1 may provide a biological
option for biohydrogen production as well as poultry
Among the genus of Fervidobacterium, F. islandicum AW-1
and F. pennivorans have been found as native-feather
degrading bacteria [13, 9]. Compared to other Fervidobacterium
strains, the genome-based approach for this extremely
thermophilic bacterium is of great importance and interest
not only for keratin degradation, but also for elucidation of
distinct amino acid and carbohydrate metabolic pathways.
Accordingly, these and related reasons led us to sequence the
whole genome of F. islandicum AW-1, providing an insight
into the degradation of non-digestible keratin biomass.
Moreover, comparative genomics for feather-degrading
F. islandicum AW-1 and its closely related
nondegrading bacteria will shed light on the evolutionary
relationships among them. Overall, this genomic
analysis may provide not only an insight into the
mechanism of keratin degradation, but also an industrial option
applicable for the treatment of non-digestible biomass.
RAST: Rapid Annotation using Subsystem Technology; iPath: Interactive
Pathway Explorer; KEGG: Kyoto Encyclopedia of Genes and Genomes.
The authors declare that they have no competing interests.
YJL, HJ and SJL drafted the manuscript, performed laboratory experiments,
and analysed the data. YJL, SJL, MKP, and JYK cultured samples and
performed the electron micrograph and phylogenetic analysis. GSP, HJ, YK,
and JHS sequenced, assembled, and annotated the genome. DWL, YJL, SJL,
JHS, and KHK conceived of the study, and participated in its design and
coordination and helped to draft the manuscript. All authors read and
approved the final manuscript.
We thank to Young Mi Sim (KOBIC) for technical assistance. This work
was supported by a grant for Agricultural R&D (PJ009767) from the Rural
Development Administration in Korea.
1. Martinez-Hernandez A-L , Velasco-Santos C , De-Icaza M , Castaño V. Hierarchical Microstructure in Keratin Biofibers . Microsc Microanal . 2003 ; 9 (SupplementS02): 1282 - 3 .
2. Poole AJ , Church JS , Huson MG . Environmentally sustainable fibers from regenerated protein . Biomacromolecules . 2009 ; 10 ( 1 ): 1 - 8 .
3. Williams CM . Development of environmentally superior technologies in the US and policy . Bioresour Technol . 2009 ; 100 ( 22 ): 5512 - 8 .
4. Bertsch A , Coello N. A biotechnological process for treatment and recycling poultry feathers as a feed ingredient . Bioresour Technol . 2005 ; 96 ( 15 ): 1703 - 8 .
5. Gushterova A , Vasileva-Tonkova E , Dimova E , Nedkov P , Haertlé T . Keratinase Production by Newly Isolated Antarctic Actinomycete Strains. World J Microbiol Biotechnol . 2005 ; 21 ( 6-7 ): 831 - 4 .
6. Williams CM , Richter CS , Mackenzie JM , Shih JC . Isolation, identification, and characterization of a feather-degrading bacterium . Appl Environ Microbiol . 1990 ; 56 ( 6 ): 1509 - 15 .
7. Santos R , Firmino AA , de Sa CM , Felix CR . Keratinolytic activity of Aspergillus fumigatus fresenius . Curr Microbiol . 1996 ; 33 ( 6 ): 364 - 70 .
8. Bockle B , Galunsky B , Muller R. Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530 . Appl Environ Microbiol . 1995 ; 61 ( 10 ): 3705 - 10 .
9. Nam GW , Lee DW , Lee HS , Lee NJ , Kim BC , Choe EA , et al. Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe . Arch Microbiol . 2002 ; 178 ( 6 ): 538 - 47 .
10. Huber R , Langworthy TA , Konig H , Thomm H , Woese CR , Sleytr UB , et al. Thermotoga maritima sp. nov . represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch Microbiol . 1986 ; 144 : 324 - 33 .
11. Huber R , Woese CR , Langworthy TA , Kristjansson JK , Stetter KO . Fervidobacterium islandicum sp. nov., a new extremely thermophilic eubacterium belonging to the "Thermotogales" . Arch Microbiol . 1990 ; 154 : 105 - 11 .
12. Andrews KT , Patel BK . Fervidobacterium gondwanense sp. nov., a new thermophilic anaerobic bacterium isolated from nonvolcanically heated geothermal waters of the Great Artesian Basin of Australia . Int J Syst Bacteriol . 1996 ; 46 ( 1 ): 265 - 9 .
13. Friedrich AB , Antranikian G . Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales . Appl Environ Microbiol . 1996 ; 62 ( 8 ): 2875 - 82 .
14. Cai J , Wang Y , Liu D , Zeng Y , Xue Y , Ma Y , et al. Fervidobacterium changbaicum sp. nov., a novel thermophilic anaerobic bacterium isolated from a hot spring of the Changbai Mountains , China. Int J Syst Evol Microbiol . 2007 ; 57 (Pt 10): 2333 - 6 .
15. Podosokorskaya OA , Merkel AY , Kolganova TV , Chernyh NA , Miroshnichenko ML , Bonch-Osmolovskaya EA , et al. Fervidobacterium ripariumsp . nov. , a thermophilic anaerobic cellulolytic bacterium isolated from a hot spring . Int J Syst Evol Microbiol . 2011 ; 61 (Pt 11): 2697 - 701 .
16. Patel BKC , Morgan HW , Daniel RM. Fervidobacterium nodosum gen . nov. and spec . nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium. Arch Microbiol . 1985 ; 141 : 63 - 9 .
17. Aziz RK , Bartels D , Best AA , DeJongh M , Disz T , Edwards RA , et al. The RAST Server: rapid annotations using subsystems technology . BMC Genomics . 2008 ; 9 : 75 .
18. Richter M , Rossello-Mora R. Shifting the genomic gold standard for the prokaryotic species definition . Proc Natl Acad Sci U S A . 2009 ; 106 ( 45 ): 19126 - 31 .
19. Chin CS , Alexander DH , Marks P , Klammer AA , Drake J , Heiner C , et al. Nonhybrid , finished microbial genome assemblies from long-read SMRT sequencing data . Nat Methods . 2013 ; 10 ( 6 ): 563 - 9 .
20. Tatusov RL , Galperin MY , Natale DA , Koonin EV . The COG database: a tool for genome-scale analysis of protein functions and evolution . Nucleic Acids Res . 2000 ; 28 ( 1 ): 33 - 6 .
21. Eid J , Fehr A , Gray J , Luong K , Lyle J , Otto G , et al. Real-time DNA sequencing from single polymerase molecules . Science . 2009 ; 323 ( 5910 ): 133 - 8 .
22. Besemer J , Lomsadze A , Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions . Nucleic Acids Res . 2001 ; 29 ( 12 ): 2607 - 18 .
23. Tatusova TDM , Badretdin A , Chetvernin V , Ciufo S , Li W. Prokaryotic Genome Annotation Pipeline . In: The NCBI Handbook [Internet]. 2nd ed. Bethesda (MD): National Center for Biotechnology Information (US) ; 2013 .
24. Finn RD , Bateman A , Clements J , Coggill P , Eberhardt RY , Eddy SR , et al. Pfam: the protein families database . Nucleic Acids Res . 2014 ; 42 (Database issue): D222 - 30 .
25. Petersen TN , Brunak S , von Heijne G , Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods . 2011 ; 8 ( 10 ): 785 - 6 .
26. Krogh A , Larsson B , von Heijne G , Sonnhammer ELL . Predicting transmembrane protein topology with a hidden markov model: application to complete genomes . J Mol Biol . 2001 ; 305 ( 3 ): 567 - 80 .
27. Conesa A , Gotz S , Garcia-Gomez JM , Terol J , Talon M , Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research . Bioinformatics . 2005 ; 21 ( 18 ): 3674 - 6 .
28. Markowitz VM , Mavromatis K , Ivanova NN , Chen IM , Chu K , Kyrpides NC . IMG ER: a system for microbial genome annotation expert review and curation . Bioinformatics . 2009 ; 25 ( 17 ): 2271 - 8 .
29. Field D , Garrity G , Gray T , Morrison N , Selengut J , Sterk P , et al. The minimum information about a genome sequence (MIGS) specification . Nat Biotech . 2008 ; 26 ( 5 ): 541 - 7 .
30. Woese CR , Kandler O , Wheelis ML . Towards a natural system of organisms: proposal for the domains Archaea , Bacteria, and Eucarya . Proc Natl Acad Sci . 1990 ; 87 ( 12 ): 4576 - 9 .
31. Bhandari V , Gupta R. Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord . nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam . nov., Kosmotogaceae fam. nov. and Petrotogaceae fam . nov.) and a new genus Pseudothermotoga gen . nov. with five new combinations . Antonie Van Leeuwenhoek . 2014 ; 105 ( 1 ): 143 - 68 .
32. Reysenbach A. Phylum BII . Thermotogae phy . nov. In: Garrity GM, Boone DR , Castenholz RW, editors. Bergey's Manual of Systematic Bacteriology , Second Edition , Volume 1 . New York : Springer; 2001 . p. 369 .
33. Reysenbach AL . Class I. Thermotogae class . nov. In: Garrity GM, Boone DR , Castenholz RW, editors. Bergey's Manual of Systematic Bacteriology , Second Edition , Volume 1 . New York : Springer; 2001 . p. 369 - 87 .
34. Reysenbach AL . Order I. Thermotogales ord . nov. Huber and Stetter 1992c , 3809. In: Garrity GM, Boone DR , Castenholz RW, editors. Bergey's Manual of Systematic Bacteriology , vol. Volume 1. Second Editionth ed. New York : Springer; 2001 . p. 369 .
35. Ashburner M , Ball CA , Blake JA , Botstein D , Butler H , Cherry JM , et al. Gene Ontology: tool for the unification of biology . Nat Genet . 2000 ; 25 ( 1 ): 25 - 29 .