Standards in Genomic Sciences (2012) 6:293-303
DOI:10.4056/sigs.2736042
Complete genome sequence of the moderately
thermophilic mineral-sulfide-oxidizing firmicute
Sulfobacillus acidophilus type strain (NALT)
Iain Anderson1, Olga Chertkov1,2, Amy Chen3, Elizabeth Saunders1,2, Alla Lapidus1, Matt
Nolan1, Susan Lucas1, Nancy Hammon1, Shweta Deshpande1, Jan-Fang Cheng1, Cliff Han1,2,
Roxanne Tapia1,2, Lynne A. Goodwin1,2, Sam Pitluck1, Konstantinos Liolios1, Ioanna Pagani1,
Natalia Ivanova1, Natalia Mikhailova1, Amrita Pati1, Krishna Palaniappan3, Miriam Land1,4,
Chongle Pan1,4, Manfred Rohde5, Rüdiger Pukall6, Markus Göker6, John C. Detter1,2, Tanja
Woyke1, James Bristow1, Jonathan A. Eisen1,7, Victor Markowitz3, Philip Hugenholtz1,8,
Nikos C. Kyrpides1, Hans-Peter Klenk6*, and Konstantinos Mavromatis1
1
DOE Joint Genome Institute, Walnut Creek, California, USA
Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA
3
Biological Data Management and Technology Center, Lawrence Berkeley National
Laboratory, Berkeley, California, USA
4
Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
5
HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany
6
Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany
7
University of California Davis Genome Center, Davis, California, USA
8
Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The
University of Queensland, Brisbane, Australia
2
*Corresponding author: Hans-Peter Klenk (hpk@dsmz.de)
Keywords: aerobic, motile, Gram-positive, acidophilic, moderately thermophilic, sulfide- and
iron-oxidizing, biomining, autotrophic, mixotrophic, soil, insertis sedis, Clostridiales, GEBA
Sulfobacillus acidophilus Norris et al. 1996 is a member of the genus Sulfobacillus which
comprises five species of the order Clostridiales. Sulfobacillus species are of interest for comparison to other sulfur and iron oxidizers and also have biomining applications. This is the
first completed genome sequence of a type strain of the genus Sulfobacillus, and the second
published genome of a member of the species S. acidophilus. The genome, which consists of
one chromosome and one plasmid with a total size of 3,557,831 bp harbors 3,626 proteincoding and 69 RNA genes, and is a part of the Genomic Encyclopedia of Bacteria and
Archaea project.
Introduction
The genus Sulfobacillus currently consists of five
species [1], all of which are mildly thermophilic or
thermotolerant acidophiles [2]. Sulfobacilli grow
mixotrophically by oxidizing ferrous iron, sulfur, and
mineral sulfides in the presence of yeast extract or
other organic compounds [3]. Some can also grow
autotrophically [2,3]. The strains that have been
tested are capable of anaerobic growth using Fe+3 as
an electron acceptor [2,4]. The genus Sulfobacillus,
along with the genus Thermaerobacter, have only
tentatively been assigned to a family, “Clostridiales
Family XVII incertae sedis”. This group may form a
deep branch within the phylum Firmicutes or may
constitute a new phylum [5]. Strain NALT (= DSM
10332 = ATCC 700253) is the type strain of the species Sulfobacillus acidophilus. The genus name was
derived from the Latin words 'sulfur' and 'bacillus'
meaning 'small sulfur-oxidizing rod' [6]. The species
epithet is derived from the Neo-Latin words
'acidum', acid, and 'philus', loving, meaning acidloving [3]. The first genome from a member of the
species S. acidophilus, strain TPY, which was isolated
The Genomic Standards Consortium
Sulfobacillus acidophilus type strain (NALT)
from a hydrothermal vent in the Pacific Ocean, was
recently sequenced by Li et al. [7]. Here we present a
summary classification and a set of features for S.
acidophilum strain NALT, together with the description of the complete genomic sequencing and annotation.
Classification and features
A representative genomic 16S rRNA sequence of S.
acidophilus NALT was compared using NCBI
BLAST [8,9] under default settings (e.g., considering only the high-scoring segment pairs (HSPs)
from the best 250 hits) with the most recent release of the Greengenes database [10] and the relative frequencies of taxa and keywords (reduced
to their stem [11]) were determined, weighted by
BLAST scores. The most frequently occurring genera were Sulfobacillus (81.9%), Thermaerobacter
(8.0%), Laceyella (2.8%), 'Gloeobacter' (2.1%) and
'Synechococcus' (2.0%) (76 hits in total). Regarding the six hits to sequences from members of the
species, the average identity within HSPs was
98.9%, whereas the average coverage by HSPs
was 97.2%. Regarding the 23 hits to sequences
from other members of the genus, the average
identity within HSPs was 93.1%, whereas the average coverage by HSPs was 81.2%. Among all
other species, the one yielding the highest score
was “Sulfobacillus yellowstonensis” (AY007665),
which corresponded to an identity of 99.4% and
an HSP coverage of 97.0%. (Note that the
Greengenes database uses the INSDC (=
EMBL/NCBI/DDBJ) annotation, which is not an
authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was HQ730681 ('Microbial Anaerobic Sediments Tinto River: Natural Acid and Heavy Metals
Content extreme acid clone SN1 2009 12D'),
which showed an identity of 94.5% and an HSP
coverage of 99.0%. The most frequently occurring
keywords within the labels of all environmental
samples which yielded hits were 'acid' (4.8%),
'soil' (4.5%), 'hydrotherm' (3.7%), 'microbi'
(3.7%) and 'mine' (3.0%) (172 hits in total). These
keywords correspond well to the environment
from which strain NALT was isolated. Environmental samples that yielded hits of a higher score
than the highest scoring species were not found.
Figure 1 shows the phylogenetic neighborhood of
S. acidophilus NALT in a 16S rRNA based tree. The
sequences of the five 16S rRNA gene copies in the
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genome differ from each other by up to eight nucleotides, and differ by up to four nucleotides from
the previously published 16S rRNA sequence
(AB089842), which contains two ambiguous base
calls.
Cells of S. acidophilus NALT are rods 3.0-5.0 μm in
length and 0.5-0.8 μm in width (Table 1 and Figure 2) [3]. Cells are Gram-positive and form spherical endospores [3]. Flagella were not observed
[3]. Strain NALT was found to grow between 28°C
and 62°C with an optimum at 48°C [35]. The upper and lower temperatures for growth were not
determined but were predicted to be 10°C and
62°C [35]. The pH range for growth was 1.6-2.3
with an optimum at 1.8 [35]. Three strains of S.
acidophilus have been found to be facultative anaerobes that are able to use Fe+3 as an electron
acceptor under anaerobic conditions [4]; but
strain NALT was not tested in this study. Strain
NALT can grow autotrophically or mixotrophically
by oxidizing Fe+2, sulfur, or mineral sulfides or
heterotrophically on yeast extract [3]. S. acidophilus and other sulfobacilli have potential applications in biomining. Strain NALT increased the
leaching of numerous mineral sulfides [35], however, its sensitivity to low concentrations of metals may limit its usefulness in biomining [35].
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the
basis of its phylogenetic position [38], and is part
of the Genomic Encyclopedia of Bacteria and
Archaea project [39]. The genome project is deposited in the Genomes OnLine Database [18] and
the complete genome sequence is deposited in
GenBank. Sequencing, 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
S. acidophilus strain NALT, DSM 10332, was grown
in DSMZ medium 709 (Acidomicrobium medium)
[40] at 45°C. DNA was isolated from 0.5-1 g of cell
paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the
standard protocol as recommended by the manufacturer with modification st/LALM for cell lysis
as described in Wu et al. 2009 [39]. DNA is available through the DNA Bank Network [41].
Standards in Genomic Sciences
Anderson et al.
Figure 1. Phylogenetic tree highlighting the position of S. acidophilus relative to the type strains of the other species
within the genus Sulfobacillus. The tree was inferred from 1,422 aligned characters [12,13] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [14]. The comparatively closely related genus Symbiobacterium
[15] was included for rooting the tree. The branches are scaled in terms of the expected number of substitutions per
site. Numbers adjacent to the branches, if any, are support values from 1,000 ML bootstrap replicates [16] (left) and
from 1,000 maximum parsimony bootstrap replicates [17] (right) if larger than 60% (i.e., there were none). Lineages
with type strain genome sequencing projects registered in GOLD [18] are labeled with one asterisk, those also listed as
'Complete and Published' with two asterisks [19].
Figure 2. Scanning electron micrograph of S. acidophilus NALT
http://standardsingenomics.org
295
Sulfobacillus acidophilus type strain (NALT)
Table 1. Classification and general features of S. acidophilus NALT according to the MIGS recommendations [20] and the NamesforLife database [21].
MIGS ID
Property
Term
Evidence code
Domain Bacteria
TAS [22]
Phylum “Firmicutes”
TAS [23-25]
Class Clostridia
TAS [26,27]
Order Clostridiales
TAS [28,29]
Family “XVII incertae sedis”
TAS [5,30]
Genus Sulfobacillus
TAS [31-33]
Species Sulfobacillus acidophilus
TAS [3,34]
Type strain NAL
TAS [3]
Gram stain
positive
TAS [3]
Cell shape
rods
TAS [3]
Motility
non-motile
NAS
Sporulation
spherical endospores
TAS [3]
Temperature range
not reported
Optimum temperature
48°C
Salinity
not reported
Oxygen requirement
facultative anaerobe
TAS [4]
Carbon source
CO2, organic compounds
TAS [3]
Energy metabolism
autotrophic, mixotrophic, heterotrophic
TAS [3]
MIGS-6
Habitat
acidic sulfidic and sulfurous sites
TAS [35]
MIGS-15
Biotic relationship
free-living
TAS [3]
MIGS-14
Pathogenicity
none
NAS
Biosafety level
1
TAS [36]
Isolation
coal spoil heap
TAS [3]
MIGS-4
Geographic location
Alvecote, North Warwickshire, UK
TAS [3]
MIGS-5
Sample collection time
1988
TAS [3]
MIGS-4.1
Latitude
52.638
TAS [3]
MIGS-4.2
Longitude
-1.641
TAS [3]
MIGS-4.3
Depth
not reported
MIGS-4.4
Altitude
not reported
Current classification
MIGS-22
TAS [35]
Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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 [37]. If the
evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements.
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Anderson et al.
Table 2. Genome sequencing project information
MIGS ID
Property
Term
MIGS-31
Finishing quality
MIGS-28
Libraries used
MIGS-29
Sequencing platforms
Illumina GAii, 454 GS FLX Titanium
MIGS-31.2
Sequencing coverage
MIGS-30
Assemblers
MIGS-32
Gene calling method
168.4 × Illumina; 51.2 × pyrosequence
Newbler version 2.3-PreRelease-6/30/2009, Velvet 1.0.13,
phrap version SPS - 4.24
Prodigal 1.4, GenePRIMP
INSDC ID
MIGS-13
Genbank Date of Release
GOLD ID
NCBI project ID
Database: IMG-GEBA
Source material identifier
Project relevance
Finished
Four genomic libraries: one 454 pyrosequence standard library,
two 454 PE libraries (6 kb and 10 kb insert size), one Illumina library
CP003179 (chromosome)
CP003180 (plasmid, unnamed)
December 14, 2011
Gc02053
40777
2506520015
DSM 10332
Tree of Life, GEBA, biomining
Genome sequencing and assembly
The genome was sequenced using a combination of
Illumina and 454 sequencing platforms. All general
aspects of library construction and sequencing can be
found at the JGI website [42]. Pyrosequencing reads
were assembled using the Newbler assembler
(Roche). The initial Newbler assembly consisting of
104 contigs in three scaffolds was converted into a
phrap [43] assembly by making fake reads from the
consensus, to collect the read pairs in the 454 paired
end library. Illumina GAii sequencing data (599.7 Mb)
were assembled with Velvet [44] and the consensus
sequences were shredded into 1.5 kb overlapped fake
reads and assembled together with the 454 data. The
454 draft assembly was based on 143.7 Mb of 454
draft data and all of the 454 paired-end data. Newbler
parameters were -consed -a 50 -l 350 -g -m -ml 20.
The Phred/Phrap/Consed software package [43] was
used for sequence assembly and quality assessment
in the subsequent finishing process. After the shotgun
stage, reads were assembled with parallel phrap
(High Performance Software, LLC). Possible misassemblies were corrected with gapResolution (C.
Han, unpublished), Dupfinisher [45], or sequencing
cloned bridging PCR fragments with subcloning. Gaps
between contigs were closed by editing in Consed,
PCR and Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 640 additional reactions and
eight shatter libraries were necessary to close gaps
and to raise the quality of the finished sequence.
Illumina reads were also used to correct potential
base errors and increase consensus quality using the
software Polisher developed at JGI [46]. The error
rate of the completed genome sequence is less than 1
http://standardsingenomics.org
in 100,000. Together, the combination of the Illumina
and 454 sequencing platforms provided 219.6 × coverage of the genome. The final assembly contained
612,059 pyrosequence and 16,626,072 Illumina
reads.
Genome annotation
Genes were identified using Prodigal [47] as part of
the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual
curation using the JGI GenePRIMP pipeline [48].
The predicted CDSs were translated and used to
search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt,
TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro
databases. Additional gene prediction analysis and
functional annotation was performed within the
Integrated Microbial Genomes - Expert Review
(IMG-ER) platform [49].
Genome properties
The genome consists of one circular chromosome of
3,472,898 bp and one circular plasmid of 84,933 bp
length with an overall G+C content of 56.8% (Table 3
and Figures 3 and 4). Based on coverage of 454
paired ends, the plasmid may be inserted into the
chromosome in about half of the population. Of the
3,695 genes predicted, 3,626 are protein-coding
genes, and 69 are RNAs; 155 pseudogenes were also
identified. The majority of the protein-coding genes
(68.3%) were assigned a putative function while the
remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional
categories is presented in Table 4.
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Sulfobacillus acidophilus type strain (NALT)
Table 3. Genome Statistics
Attribute
Value
% of Totala
Genome size (bp)
3,557,831
100.00%
DNA coding region (bp)
3,106,298
87.31%
DNA G+C content (bp)
2,019,235
56.75%
Number of replicons
2
Extrachromosomal elements
1
Total genes
3,695
RNA genes
69
rRNA operons
Protein-coding genes
5
3,626
100.00%
155
4.27%
Genes with function prediction
2,475
68.26%
Genes in paralog clusters
1,896
52.29%
Genes assigned to COGs
2,740
75.57%
Genes assigned Pfam domains
413
11.39%
Genes with signal peptides
652
17.98%
Genes with transmembrane helices
910
25.10%
Pseudo genes
CRISPR repeats
2
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.
Insights into the genome sequence
Comparative genomics
While the sequencing of the genome described in
this paper was underway, Li et al. from the Third
Institute of Oceanography, Xiamen, China published the complete genome sequence of strain
TPY [7]. The two genomes differ in size by less
than 7,000 bp. Here, we take the opportunity to
compare the completed genome sequences from
these two stains, NALT and TPY, both belonging to
S. acidophilus. While the biological material for the
type stain, NALT, is publicly available from the
DSMZ open collection for postgenomic analyses,
no source of the biological material (MIGS-13 criterion, see Table 2) of strain TPY was provided by
Li et al. [7].
To estimate the overall similarity between the genomes of strains NALT and TPY (Genbank accession number: CP002901), the GGDC-Genome-toGenome Distance Calculator [50,51] was used. The
system calculates the distances by comparing the
298
genomes to obtain HSPs (high-scoring segment
pairs) and interfering distances from three formulae (HSP length / total length; identities / HSP
length; identities / total length). The comparison
of the genomes of strains NALT and TPY revealed
that 99.65% of the average of the genome lengths
are covered with HSPs. The identity within these
HSPs was 99.01%, whereas the identity over the
whole genome (counting regions not covered by
HSPs as non-identical) was 98.67%. The inferred
digital DNA-DNA hybridization values for the two
strains are 96.47% (formula 1 in [51]), 86.08%
(formula 2 in [51]) and 97.05% (formula 3 in
[51]), respectively. These results clearly demonstrate that according to the whole genome sequences of strains NALT and TPY, the similarity is
very high, supporting the membership of both
strains in the same species.
Standards in Genomic Sciences
Anderson et al.
Figure 3. Graphical map of the chromosome. From outside to the center: Genes on forward
strand (colored by COG categories), Genes on reverse strand (colored by COG categories),
RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.
Figure 4. Graphical map of the plasmid. From outside
to the center: Genes on forward strand (colored by
COG categories), Genes on reverse strand (colored by
COG categories), RNA genes (tRNAs green, rRNAs
red, other RNAs black), GC content, GC skew.
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Sulfobacillus acidophilus type strain (NALT)
Table 4. Number of genes associated with the general COG functional categories
Code value
%agea Description
J
149
4.1 Translation, ribosomal structure and biogenesis
A
0
0.0 RNA processing and modification
K
188
5.2 Transcription
L
269
7.4 Replication, recombination and repair
B
1
0.0 Chromatin structure and dynamics
D
26
0.7 Cell cycle control, cell division, chromosome partitioning
Y
0
0.0 Nuclear structure
V
34
0.9 Defense mechanisms
T
111
3.1 Signal transduction mechanisms
M
149
4.1 Cell wall/membrane/envelope biogenesis
N
47
1.3 Cell motility
Z
0
0.0 Cytoskeleton
W
0
0.0 Extracellular structures
U
62
1.7 Intracellular trafficking, secretion, and vesicular transport
O
129
3.6 Posttranslational modification, protein turnover, chaperones
C
244
6.7 Energy production and conversion
G
215
5.9 Carbohydrate transport and metabolism
E
257
7.1 Amino acid transport and metabolism
F
89
2.5 Nucleotide transport and metabolism
H
153
4.2 Coenzyme transport and metabolism
I
130
3.6 Lipid transport and metabolism
P
121
3.3 Inorganic ion transport and metabolism
Q
81
2.2 Secondary metabolites biosynthesis, transport and catabolism
R
326
9.0 General function prediction only
S
239
6.6 Function unknown
886
24.4 Not in COGs
a) The percentage is based on the total number of protein coding genes in the annotated
genome.
The comparison of the number of genes belonging
to the different COG categories revealed few differences between the genomes of strains NALT and
TPY. Strain NALT has 2,740 genes with COGs assigned, while strain TPY has 2,700. We analyzed the
differences in COG assignment between the two
strains and found that in almost all cases they could
be explained by differences in the gene calls or
pseudogene assignment, i.e. in one genome two
parts of a pseudogene were called as two separate
genes, while in the other genome they were combined into one pseudogene. The only clear case of a
difference in gene content between the two strains
is the presence of a transposable element consisting of two genes (Sulac_1668, Sulac_1669) disrupting a subunit of a potassium transporter
(Sulac_1667) in strain NALT. There were also cases
where a gene in one strain was split into two genes
in the other strain. For example, Sulac_2178 corresponds to TPY_1983 and TPY1984, and Sulac_0347
corresponds to TPY_0381 and TPY_0382. In both
cases the differences are due to a single base indel.
300
A dot plot showed that there are large blocks of
synteny between the two genomes with some rearrangements (data not shown). The genes found on
the plasmid in strain NALT are found in two regions
of the chromosome in strain TPY. Sulac_3528-3555
corresponds to TPY_0524-0552, while Sulac_35563626 corresponds to TPY_2310-2244. This suggests that in strain TPY, the plasmid was inserted
into the chromosome and then split into two pieces.
We analyzed CRISPR repeats with the CRISPR
Recognition Tool [52] and found major differences
between the two strains. They both have two regions of CRISPR repeats, but the strain TPY repeat
regions have 8 and 9 repeats while the strain NALT
repeat regions have 27 and 43 repeats. All of the
spacers in the TPY repeat regions are found in
NALT, but NALT has many additional spacers. This
agrees with previous results suggesting that
CRISPRs evolve quickly, and differences can be
found in closely related strains [53].
Standards in Genomic Sciences
Anderson et al.
Acknowledgements
We would like to gratefully acknowledge the help of
Gabriele Gehrich-Schröter for growing S. acidophilus
cultures and Susanne Schneider for DNA extraction
(both at DSMZ). This work was performed under the
auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program,
and by the University of California, Lawrence Berkeley
National Laboratory under contract No. DE-AC02-
05CH11231, Lawrence Livermore National Laboratory
under Contract No. DE-AC52-07NA27344, and Los
Alamos National Laboratory under contract No. DEAC02-06NA25396, UT-Battelle and Oak Ridge National
Laboratory under contract DE-AC05-00OR22725, as
well as German Research Foundation (DFG) INST
599/1-2.
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