Complete genome sequence of Ferroglobus placidus AEDII12DO.

Abstract

Ferroglobus placidus belongs to the order Archaeoglobales within the archaeal phylum Euryarchaeota. Strain AEDII12DO is the type strain of the species and was isolated from a shallow marine hydrothermal system at Vulcano, Italy. It is a hyperthermophilic, anaerobic chemolithoautotroph, but it can also use a variety of aromatic compounds as electron donors. Here we describe the features of this organism together with the complete genome sequence and annotation. The 2,196,266 bp genome with its 2,567 protein-coding and 55 RNA genes was sequenced as part of a DOE Joint Genome Institute Laboratory Sequencing Program (LSP) project.

Full text

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Standards in Genomic Sciences (2011) 5:50-60
DOI:10.4056/sigs.2225018

The Genomic Standards Consortium
Complete genome sequence of Ferroglobus placidus
AEDII12DO
Iain Anderson,1* Carla Risso,2 Dawn Holmes,2 Susan Lucas,1 Alex Copeland,1 Alla Lapidus,1
Jan-Fang Cheng,1 David Bruce,1,3 Lynne Goodwin,1,3 Samuel Pitluck,1 Elizabeth Saunders,1,3
Thomas Brettin,1,3 John C. Detter,1,3 Cliff Han,1,3 Roxanne Tapia,1,3 Frank Larimer,4 Miriam
Land,4 Loren Hauser,4 Tanja Woyke,1 Derek Lovley,2 Nikos Kyrpides,1 and Natalia Ivanova1
1DOE Joint Genome Institute, Walnut Creek, California, USA
2Microbiology Department, University of Massachusetts, Amherst, Massachusetts, USA
3Los Alamos National Laboratory, Los Alamos, New Mexico, USA
4Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
*Corresponding author: IJAnderson@lbl.gov
Keywords: Archaea, Euryarchaeota, Archaeoglobales, hydrothermal vent, hyperthermophile,
anaerobe
Ferroglobus placidus belongs to the order Archaeoglobales within the archaeal phylum Euryar-
chaeota. Strain AEDII12DO is the type strain of the species and was isolated from a shallow
marine hydrothermal system at Vulcano, Italy. It is a hyperthermophilic, anaerobic chemoli-
thoautotroph, but it can also use a variety of aromatic compounds as electron donors. Here we
describe the features of this organism together with the complete genome sequence and anno-
tation. The 2,196,266 bp genome with its 2,567 protein-coding and 55 RNA genes was se-
quenced as part of a DOE Joint Genome Institute Laboratory Sequencing Program (LSP) project.
Introduction
Strain AEDII12DO (=DSM 10642) is the type strain
of the species Ferroglobus placidus. It was isolated
from a shallow hydrothermal vent system at Vulca-
no Island, Italy [1]. F. placidus is metabolically quite
versatile. It was isolated based on its ability to use
ferrous iron as an electron donor, and was also
shown to use hydrogen and sulfide as electron do-
nors, with nitrate or thiosulfate as electron accep-
tors [1]. Subsequently, it was shown to produce
N2O from nitrite, which is an unusual ability for an
anaerobic organism [2]. It can also oxidize acetate
and several aromatic compounds using ferric iron
as the electron acceptor [3,4]. F. placidus is the first
archaeon found to anaerobically oxidize aromatic
compounds [4]. The genes and pathways involved
in degradation of benzene, benzoate, and phenol
have been recently characterized [5,6].
F. placidus is the only species in the genus Ferroglo-
bus. It belongs to the family Archaeoglobaceae,
which also contains the genera Archaeoglobus and
Geoglobus. Genome sequences have been published
for A. fulgidus and A. profundus [7,8]. Figure 1
shows the phylogenetic relationships between
members of the family Archaeoglobaceae.
Organism information
F. placidus was isolated from a mixture of sand
and water at a beach close to Vulcano Island, Italy
[1]. The sample was taken from a depth of 1 m; the
temperature of the sample was 95°C and the pH
was 7.0 [1]. A 1.0 mL aliquot of the sample was
incubated in FM medium at 85°C with shaking.
The medium contained FeS as an electron donor
[1]. F. placidus was isolated from the enrichment
culture using optical tweezers [1]. The cells are
irregular cocci with a triangular shape, and one or
two flagella were present [1]. Growth occurred
between 65°C and 95°C with an optimum of 85°C
[1]. The optimal pH for growth was 7.0, and
growth was observed over a range of 6.0 to 8.5
[1]. The optimal salinity for growth was 2.0%,
with growth occurring between 0.5 and 4.5% NaCl
[1]. F. placidus could use ferrous iron, hydrogen, or
sulfide as electron donors and nitrate or thiosul-
fate as electron acceptors [1]. F. placidus also can
anaerobically oxidize aromatic compounds with
ferric iron as electron acceptor. The aromatic
compounds it can utilize include benzene, ben-
zoate, phenol, 4-hydroxybenzoate, benzaldehyde,
p-hydroxybenzaldehyde and t-cinnamic acid [4,5].
The features of the organism are listed in Table 1.

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Anderson et al.
http://standardsingenomics.org 51

Figure 1. 16S ribosomal RNA phylogenetic tree of Archaeoglobaceae. The tree was generated with weighbor [9]
through the Ribosomal Database Project website [10] and displayed with njplot [11]. Organisms with two aste-
risks after the name are those with complete genomes sequenced and published. Those with one asterisk have a
genome project in progress, according to the Genomes OnLine Database [12]. Methanocaldococcus jannaschii
is the outgroup.
Genome sequencing information
Genome project history
This organism was selected for sequencing based
on its phylogenetic position and its phenotypic dif-
ferences from other members of the family Arc-
haeoglobaceae. It is part of a Laboratory Sequenc-
ing Program (LSP) project to sequence diverse arc-
haea. The genome project is listed in the Genomes
On Line Database [12] and the complete genome
sequence has been deposited in GenBank. Sequenc-
ing, finishing, and annotation were performed by
the DOE Joint Genome Institute (JGI). A summary of
the project information is shown in Table 2.
Growth conditions and DNA isolation
The strain Ferroglobus
(containing plasmid XY) has been deposited in the
Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ) by Prof. Dr. K. O. Stetter,
Lehrstuhl für Mikrobiologie, Universität Regensburg,
Universitätsstr. 31, D-93053 Regensburg, Germany
as DSM 10642.
F. placidus strain AEDII12DO was obtained from
the DSMZ. Strict anaerobic culturing and sampling
techniques were used throughout [22,23]. Ten 100
ml bottles of F. placidus cells were grown with ace-
tate (10 mM) as the electron donor, and Fe(III) ci-
trate (56 mM) as the electron acceptor. F. placidus
medium was prepared as previously described [4].
After autoclaving, FeCl2 (1.3 mM), Na2SeO4 (30
µg/L), Na2WO4 (40 µg/L), APM salts (1 g/L MgCl2,
0.23 g/L CaCl2), DL vitamins [24] and all electron
donors were added to the sterilized medium from
placidus AEDII12DO
anaerobic stock solutions. Cultures were incubated
under N2:CO2 (80:20) at 85 °C in the dark.
For extraction of DNA, cultures (100 ml in 156 ml
serum bottles) were divided into 50 ml conical
tubes (Falcon), and cells were pelleted by centrifu-
gation at 3,000 x g for 20 minutes. Cell pellets were
resuspended in 10 ml TE sucrose buffer (10 mM
Tris, pH 8.0, 1 mM EDTA, and 6.7% sucrose). The
resuspended cells were distributed into 10 differ-
ent 2 ml screw cap tubes and 3 µl Proteinase K (20
mg/ml), 30 µl sodium dodecyl sulfate (10% solu-
tion), and 10 µl RNase A (5 ug/ul) were added to
each tube. Tubes were incubated at 37ºC for 20
min, and centrifuged at 16,100 x g for 15 minutes.
The supernatant was transferred to a new set of
tubes and 600 µl phenol (TE saturated, pH 7.3), and
400 μl chloroform-isoamyl alcohol were added.
These tubes were then mixed on a Labquake rota-
tor (Barnstead/Thermolyne, Dubuque, Iowa) for 10
min and centrifuged at 16,100 x g for 5 min. The
aqueous layer was removed and transferred to new
2-ml screw cap tubes. The phenol/chloroform ex-
traction step was performed again. The aqueous
layer was transferred to a new tube, and 100 µl 5 M
ammonium acetate, 20 µl glycogen (5 mg/ml; Am-
bion), and 1 ml cold (-20 °C) isopropanol (Sigma)
were added. Nucleic acids were precipitated at -30
°C for 1 hour and pelleted by centrifugation at
16,100 x g for 30 min. The pellet was then cleaned
with cold (-20 °C) 70% ethanol, dried, and resus-
pended in sterile nuclease-free water (Ambion).

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Ferroglobus placidus AEDII12DO
52 Standards in Genomic Sciences
Table 1. Classification and general features of F. placidus AEDII12DO according to the MIGS recommendations [13].
MIGS ID Property Term

Current classification
Domain Archaea

Phylum Euryarchaeota

Class Archaeoglobi

Order Archaeoglobales

Family Archaeoglobaceae

Genus Ferroglobus

Species Ferroglobus placidus

Type strain AEDII12DO

Cell shape irregular coccus

Motility motile

Sporulation nonsporulating

Temperature range 65-95°C

Optimum temperature 85°C
MIGS-6.3 Salinity 0.5-4.5% NaCl (optimum 2%)
MIGS-22 Oxygen requirement anaerobe

Carbon source CO2

Energy metabolism chemolithotrophic, chemoorganotrophic
MIGS-6 Habitat marine geothermally heated areas
MIGS-15 Biotopic relationship free-living
MIGS-14 Pathogenicity none

Biosafety level 1

Isolation geothermally heated sediment
MIGS-4 Geographic location Vulcano island, Italy
MIGS-5 Isolation time unknown
MIGS-4.1 Latitude 38.4154
MIGS-4.2 Longitude 14.9609
MIGS-4.3 Depth 1 m
MIGS-4.4 Altitude not applicable
Evidence codea
TAS [14]
TAS [15]
TAS [16,17]
TAS [17,18]
TAS [17,19]
TAS [1,20]
TAS [1,20]
TAS [1]
TAS [1]
TAS [1]
NAS
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1,3,4]
TAS [1]
TAS [1]
NAS
NAS
TAS [1]
TAS [1]

TAS [1]
TAS [1]
TAS [1]

a) Evidence 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 [21].
Genome sequencing and assembly
The genome of F. placidus was sequenced at the
Joint Genome Institute using a combination of Il-
lumina and 454 technologies. An Illumina GAII
shotgun library with reads of 539 Mb, a 454 Tita-
nium draft library with average read length of
292.3 bases, and a paired-end 454 library with an
average insert size of 15.5 Kb were generated for
this genome. All general aspects of library con-
struction and sequencing performed at the JGI can
be found at the DOE JGI website [25].
Illumina sequencing data was assembled with
Velvet [26], and the consensus sequences were
shredded into 1.5 kb overlapped fake reads and
assembled together with the 454 data. Draft as-
semblies were based on 104 Mb 454 draft data
and 454 paired end data. The initial Newbler as-
sembly contained 33 contigs in 1 scaffold. We
converted the initial 454 assembly into a phrap
assembly by making fake reads from the consen-
sus, collecting the read pairs in the 454 paired end
library.

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Anderson et al.
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The Phred/Phrap/Consed software package [27]
was used for sequence assembly and quality as-
sessment [28-30] in the following finishing
process. After the shotgun stage, reads were as-
sembled with parallel phrap (High Performance
Software, LLC). Possible mis-assemblies were
corrected with gapResolution (Cliff Han, unpub-
lished), Dupfinisher [31], or sequencing cloned
bridging PCR fragments with subcloning or
transposon bombing (Epicentre Biotechnologies,
Madison, WI). Gaps between contigs were closed
by editing in Consed, by PCR and by Bubble PCR
primer walks. A total of 43 additional reactions
were necessary to close gaps and to raise the
quality of the finished sequence.
Genome annotation
Genes were identified using Prodigal [32], fol-
lowed by a round of manual curation using Ge-
nePRIMP [33]. The predicted CDSs were trans-
lated and used to search the National Center for
Biotechnology Information (NCBI) nonredundant
database, UniProt, TIGRFam, Pfam, PRIAM, KEGG,
COG, and InterPro databases. The tRNAScanSE
tool [34] was used to find tRNA genes, whereas
ribosomal RNAs were found by using BLASTn
against the ribosomal RNA databases. The RNA
components of the protein secretion complex and
the RNase P were identified by searching the ge-
nome for the corresponding Rfam profiles using
INFERNAL [35]. Additional gene prediction anal-
ysis and manual functional annotation was per-
formed within the Integrated Microbial Genomes
(IMG) platform [36] developed by the Joint Ge-
nome Institute, Walnut Creek, CA, USA [37].
Genome properties
The genome includes one circular chromosome
and no plasmids, for a total size of 2,196,266 bp
(Table 3 and Figure 2). This genome size is al-
most the same as that of A. fulgidus and approx-
imately 0.6 Mbp larger than that of A. profundus.
The mol percent G+C is 44.1%, close to the values
found in the Archaeoglobus genomes. A total of
2,622 genes were identified, 55 RNA genes and
2,567 protein-coding genes. There are 87 pseu-
dogenes, comprising 3.4% of the protein-coding
genes. The start codon is ATG in 83.7% of the
genes, GTG in 12.2%, and TTG in 5.8%. This dis-
tribution is more similar to that of A. profundus,
to which F. placidus is closely related (Figure 1),
than to that of A. fulgidus. There is one copy of
each ribosomal RNA. The 5S rRNA is not found
adjacent to the 16S and 23S rRNAs. Table 4
shows the distribution of genes in COG catego-
ries.
Table 2. Genome sequencing project information
MIGS ID Property Term
MIGS-31 Finishing quality Finished
Illumina standard library, 454 standard library,
454 15 kb paired end library
MIGS-28 Libraries used
MIGS-29 Sequencing platforms Illumina GA II, 454 GS FLX Titanium
MIGS-31.2 Sequencing coverage Illumina 245×, 454 47×
MIGS-30 Assemblers Velvet, Newbler, phrap
MIGS-32 Gene calling method Prodigal, GenePRIMP

INSDC ID CP001899

Genbank Date of Release February 16, 2010

GOLD ID Gc01209

NCBI project ID 33635
MIGS-13 Source material identifier DSM 10642

Project relevance Phylogenetic diversity, biotechnology

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Ferroglobus placidus AEDII12DO
54 Standards in Genomic Sciences
Table 3. Nucleotide content and gene count levels of the genome
Attribute
Size (bp)
G+C content (bp)
Coding region (bp)
Number of replicons
Extrachromosomal elements
Total genes
RNA genes
rRNA operons
Protein-coding genes
Pseudogenes
Genes with function prediction
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
CRISPR repeats
Value % of totala
100.0%
44.1%
90.9%
2,196,266
969,331
1,996,425
1
0





2,622
55
1
2,567
87
1,619
350
1,820
1,889
269
502
100.0%
3.4%
63.1%
13.6%
70.9%
73.6%
10.5%
19.6%
100.0% 6
a) The total is based on either the size of the genome in base pairs or
the total number of protein coding genes in the annotated genome.
Table 4. Number of genes associated with the 25 general COG functional categories
Code Value %agea Description
J 163 6.3% Translation
A 2 0.1% RNA processing and modification
K 90 3.5% Transcription
L 110 4.3% Replication, recombination and repair
B 8 0.3% Chromatin structure and dynamics
D 23 0.9% Cell cycle control, mitosis and meiosis
Y 0 0.0% Nuclear structure
V 16 0.6% Defense mechanisms
T 37 1.4% Signal transduction mechanisms
M 42 1.6% Cell wall/membrane biogenesis
N 24 0.9% Cell motility
Z 0 0.0% Cytoskeleton
W 0 0.0% Extracellular structures
U 27 1.1% Intracellular trafficking and secretion
O 88 3.4% Posttranslational modification, protein turnover, chaperones
C 207 8.1% Energy production and conversion
G 44 1.7% Carbohydrate transport and metabolism
E 147 5.7% Amino acid transport and metabolism
F 50 1.9% Nucleotide transport and metabolism
H 118 4.6% Coenzyme transport and metabolism
I 76 3.0% Lipid transport and metabolism
P 94 3.7% Inorganic ion transport and metabolism
Q 22 0.9% Secondary metabolites biosynthesis, transport and catabolism
R 293 11.4% General function prediction only
S 246 9.6% Function unknown
- 747 29.1% Not in COGs
a) The total is based on the total number of protein coding genes in the genome