JOURNAL OF BACTERIOLOGY, Apr. 2011, p. 1672–1680
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 7
Genome Analyses of Icelandic Strains of Sulfolobus islandicus, Model
Organisms for Genetic and Virus-Host Interaction Studies?
Li Guo,1† Kim Bru ¨gger,2† Chao Liu,2† Shiraz A. Shah,2† Huajun Zheng,3Yongqiang Zhu,3
Shengyue Wang,3Reidun K. Lillestøl,2Lanming Chen,2Jeremy Frank,2David Prangishvili,4
Lars Paulin,5Qunxin She,2‡ Li Huang,1‡* and Roger A. Garrett2‡*
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road,
Chaoyang District, Beijing 100101, China1; Archaea Centre, Department of Biology, Copenhagen University, Ole Maaløes Vej 5,
DK-2200N Copenhagen, Denmark2; Shanghai MOST Key Laboratory of Disease and Health Genomics,
Chinese National Human Genome Center at Shanghai, Shanghai 201203, China3; Molecular Biology of
the Gene in Extremophiles Unit, Institut Pasteur, rue Dr Roux 25, 75724 Paris Cedex, France4; and
DNA Sequencing and Genomics Laboratory, Institute of Biotechnology, University of
Helsinki, 00790 Helsinki, Finland5
Received 10 December 2010/Accepted 16 January 2011
The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and
analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable
genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad host range for exceptional
crenarchaeal viruses and conjugative plasmids and was selected for studying their life cycles and host
interactions. The genomes of strains REY15A and HVE10/4 are 2.5 and 2.7 Mb, respectively, and each genome
carries a variable region of 0.5 to 0.7 Mb where major differences in gene content and gene order occur. These
include gene clusters involved in specific metabolic pathways, multiple copies of VapBC antitoxin-toxin gene
pairs, and in strain HVE10/4, a 50-kb region rich in glycosyl transferase genes. The variable region also
contains most of the insertion sequence (IS) elements and high proportions of the orphan orfB elements and
SMN1 miniature inverted-repeat transposable elements (MITEs), as well as the clustered regular interspaced
short palindromic repeat (CRISPR)-based immune systems, which are complex and diverse in both strains,
consistent with them having been mobilized both intra- and intercellularly. In contrast, the remainder of the
genomes are highly conserved in their protein and RNA gene syntenies, closely resembling those of other S.
islandicus and Sulfolobus solfataricus strains, and they exhibit only minor remnants of a few genetic elements,
mainly conjugative plasmids, which have integrated at a few tRNA genes lacking introns. This provides a
possible rationale for the presence of the introns.
Iceland has been a rich source of hyperthermophilic crenar-
chaea over the past 3 decades and especially of acidothermo-
philic members of the order Sulfolobales. Many Sulfolobus is-
landicus strains (“Island” is German for “Iceland”) have also
yielded many novel viruses showing varied and sometimes
unique morphologies and exceptional genome contents. These
properties are consistent with these viruses constituting an
archaeal lineage distinct from those of bacteria and eukarya,
and they have now been classified into several new viral fam-
ilies (38, 63). In addition, a family of conjugative plasmids has
been characterized, with most members deriving from Iceland,
which appear to conjugate by a mechanism unique to the
archaeal domain (18, 37).
Although the availability of genome sequences of Sulfolobus
strains and their genetic elements has yielded important in-
sights into the biology of these model crenarchaea, a major
impediment to more detailed insights has been the paucity of
robust and versatile vector-host systems for genetic studies. A
few Sulfolobus species have been successfully employed as
hosts for such systems, including Sulfolobus solfataricus strains
P1 and 98/2 (22, 58), Sulfolobus acidocaldarius (57), and S.
islandicus strain REY15A (54). To date, the genetic tools de-
veloped for the latter host are the most versatile and include
the following: (i) Sulfolobus-Escherichia coli shuttle vectors
carrying either viral or plasmid replication origins (50); (ii)
conventional and novel gene knockout methodologies (14, 62),
and (iii) a D-arabinose-inducible expression system with a lacS
reporter gene system (35). The S. islandicus system has also
been employed successfully to demonstrate the dynamic char-
acter of the clustered regular interspaced short palindromic
repeat (CRISPR)-based immune systems of Sulfolobus when
challenged with genetic elements carrying matching viral gene
and protospacers maintained under selection (20). These de-
velopments necessitated the determination of the genome se-
quence of S. islandicus strain REY15A as a prerequisite for
successful exploitation of the genetic systems.
A second Icelandic strain, S. islandicus strain HVE10/4, has
been employed as a broad laboratory host for propagating
diverse Sulfolobus viruses and conjugative plasmids (63) and
* Corresponding author. Mailing address for R. A. Garrett: Archaea
Centre, Department of Biology, Copenhagen University, Ole Maaløes
Vej 5, DK-2200N Copenhagen, Denmark. Phone: 045-353-22010. Fax:
045-353-22128. E-mail: email@example.com. Mailing address for L.
Huang: State Key Laboratory of Microbial Resources, Institute of
Microbiology, Chinese Academy of Sciences, No. 1 West Beichen
Road, Chaoyang District, Beijing 100101, China. Phone: 086-10-
64807430. Fax: 086-10-64807429. E-mail: firstname.lastname@example.org.
† These authors contributed equally.
‡ The last three authors are joint senior authors.
?Published ahead of print on 28 January 2011.
was selected for in-depth studies of their life cycles and host
interactions. This effort received added impetus with the dem-
onstration that some genetic elements show exceptional and
sometimes unique properties of their viral life cycles or con-
jugative mechanisms (3, 8, 18, 40). Therefore, the genome
sequence of S. islandicus strain HVE10/4 was also determined.
The genome sequences of two Icelandic strains, REY15A
and HVE10/4, were analyzed and compared and contrasted
with one another and with genomes of other S. solfataricus and
S. islandicus strains isolated from different geographical loca-
tions, including Naples, Italy; Kamchatka, Russia; Lassen Vol-
canic National Park; and Yellowstone National Park (44, 53).
MATERIALS AND METHODS
Genome sequencing. S. islandicus strains REY15A and HVE10/4 were colony
purified three times and cultured essentially as described earlier (11). Total DNA
was extracted from the cells using phenol-chloroform and further purified by
CsCl density-gradient centrifugation. For strain REY15A, sequencing of shotgun
libraries with a 454 GS FLX sequenator yielded 324,123 reads with 31-fold
genome coverage. For strain HVE10/4, DNA was sonicated to yield fragments in
the size range of 1.5 to 4.0 kb, and clone libraries were generated in pUC18 using
the SmaI site. Sequencing was performed on MegaBace 1000 sequenators to
yield approximately 3-fold sequence coverage, and the sequencing data were
combined with a sequencing run using a 454 FLX sequenator to yield approxi-
mately 10- to 15-fold coverage. The genome sequences were assembled using the
phred/phrap/consed package, contigs were linked by combinatorial PCR using
primers matching to each contig end, and the PCR products were sequenced to
close the gaps. Remaining ambiguous sequence regions in the genome were
identified and resolved by generating and sequencing PCR products. Both ge-
nomes were annotated automatically and refined manually.
Sequence analyses. Open reading frames (ORFs) were predicted with Glim-
mer (13). Frameshifts were detected and checked by sequencing after manual
annotation, and the remaining frameshifts were considered to be authentic.
Functional assignments of ORFs are based on searches against GenBank (http:
//www.ncbi.nlm.nih.gov/) and the Conserved Domain Database (CDD) (www
.ncbi.nlm.nih.gov/cdd/). tRNA genes were located with tRNAscan-SE (26). Po-
tential noncoding RNAs were predicted by comparison with the untranslated
RNAs characterized for S. solfataricus and S. acidocaldarius, in terms of se-
quence similarity and gene context (see Results). Putative insertion sequence
(IS) elements were identified by BLASTN search against the IS Finder database
(http://www-is.biotoul.fr/). All annotations were manually curated using Artemis
Genome general properties. Genomes of the two Icelandic
strains were sequenced using a combination of sequencing
strategies. S. islandicus REY15A was determined primarily by
454 sequencing, while strain HVE10/4 was obtained by a com-
bination of Sanger and 454 sequencing at approximately 30-
fold and 10-fold coverage, respectively. Protein-coding genes
were annotated in Artemis (47), where start codons for single
genes and first genes of Sulfolobus operons were generally
located 25 to 30 bp downstream from the archaeal hexameric
TATA-like box and only genes within operons were preceded
by Shine-Dalgarno motifs, of which GGUG predominates
(56). Where alternative start codons were juxtapositioned, we
selected the most probable on the basis of its position relative
to the putative promoter and/or Shine-Dalgarno motifs or ex-
perimental data from closely related organisms.
Dot plots of the two genomes demonstrate long sections of
gene synteny. One region of about 0.5 to 0.7 Mb exhibits
extensive gene shuffling, and there is a smaller region with a
200-kb inversion bordered by shuffled genes (Fig. 1A). Some of
the minor irregularities in the dot plot were attributable to
insertion or integration events. The synteny is maintained, to a
large degree, when each genome is compared to that of S.
solfataricus P2, despite the occurrence of a large inversion in
the latter, and this is illustrated in a dot plot for the genomes
of strain REY15A and S. solfataricus P2 (Fig. 1B). This exten-
sive gene synteny is surprising, given the high level of transpo-
sitional activity occurring in S. solfataricus (Table 1) (7, 30, 41).
A similar pattern was also observed when other pairs of S.
islandicus genomes from different geographical locations were
FIG. 1. (A) Dot plot of the two Icelandic genomes showing the approximate levels of sequence synteny. The large variable regions extend from
about 0.35 to 1.0 Mb. Transposase genes are denoted by black lines along the axes. Putative origins of replication adjacent to the cdc6 and whiP
genes are indicated with red circles, while the families of the CRISPR/Cas (I and III) and Cmr (B) modules are indicated by blue squares. (B) Dot
plot of the S. islandicus REY15A and S. solfataricus P2 genomes.
VOL. 193, 2011 GENOME ANALYSES OF ICELANDIC STRAINS OF S. ISLANDICUS1673
compared (48), consistent with a high level of conservation of
gene synteny for all the S. solfataricus and S. islandicus ge-
A phylogenetic tree derived from the available genomes
clusters together S. islandicus strains from different geograph-
ical locations (44), with S. solfataricus strains P2 and 98/2 being
more distantly related (Fig. 2 and Table 1). The nucleotide
sequence identity for the concatenated core genes of the two S.
islandicus genomes (Fig. 1A) is 99.6%, and between all the S.
islandicus genomes, it is about 99%. The relatively long
branches for individual strains (Fig. 2) arise mainly from dif-
ferences in gene content of the large variable regions (Fig. 1A).
The degree of sequence identity between the concatenated
core genes of the S. islandicus and S. solfataricus genomes is
about 90% (Fig. 2).
Three origins of chromosome replication, demonstrated ex-
perimentally for S. solfataricus and S. acidocaldarius (27, 46),
are well conserved with respect to both the DNA sequence and
flanking gene organization in both of the genomes, albeit with
the origin oriC2 being inverted relative to the genomes of S.
solfataricus P2 and S. islandicus strain YN1551 (Fig. 1B). Or-
igin oriC1 lies immediately upstream of cdc6-1, oriC2 is close to
cdc6-3, while oriC3 is positioned downstream of the whiP gene
(Fig. 1A). The two cdc6 genes and the whiP gene encode
putative replication initiators (45).
Large variable region. The genomes carry two types of vari-
able regions. The large region, constituting 20 to 25% of each
TABLE 1. Summary of genetic properties obtained from genomes of two Icelandic S. islandicus strains and other available S. solfataricus and S. islandicus strains
Genetic properties obtained from genomes ofb:
GenBank accession no.
Genome size (Mb)
Conserved genes (total, 1,679)
Unique single genes (total, 1,346)
Transporters (total, 15)
Glycosyl transferases (50-kb
Conserved noncoding RNAs
D, 2? B (B)
2? B, D
I, II (2? I)
II (2? I)
I, III (I)
aLetters and numbers in parentheses for the Cmr and CRISPR/Cas modules families (25, 48) denote the numbers and families of putatively defective modules generally lacking essential genes.
bLassen, Lassen Volcanic National Park; Yellowstone, Yellowstone National Park.
FIG. 2. Neighbor-joining tree based on a gene content matrix, in-
cluding the conserved, core, and unique genes for each available S.
islandicus and S. solfataricus genome (Table 1). The branch lengths
represent the number of differences between the strains in terms of the
presence or absence of individual genes. The data for the tree were
prepared using methods described earlier (44, 48). Only bootstrap
values below 100% for the individual branches are given.
1674GUO ET AL.J. BACTERIOL.
genome, extends approximately from positions encompassing
0.3 to 0.8 Mb and 0.3 to 1.0 Mb for strains REY15A and
HVE10/4, respectively (Fig. 1A). The other class is repre-
sented mainly by regions downstream from tRNA genes, where
integration events have occurred (Table 1; also, see below).
The large variable region contains about 60% of the potentially
transposable IS elements and most of the nonautonomous
mobile elements, as well as many degenerate copies of the
former (Fig. 1A). It carries some gene clusters, which are
present in one or more of the Sulfolobus genomes, including
operons and gene cassettes associated with metabolic path-
ways, and it contains the diverse CRISPR/Cas and Cmr mod-
ules (Table 1; also, see below). It generally lacks essential
genes; for example, no tRNA genes or replication origins are
present, and thus, it appears to constitute a region where
nonessential genes are collected, interchanged, and exchanged
intercellularly and where genetic innovation occurs.
Integration sites. tRNA gene integration events in Sulfolo-
bus genomes predominantly involve conjugative plasmids and
fuselloviruses, and these were also the genetic elements most
commonly isolated from acidic hot springs in Iceland (63).
Most integration events occur via an archaea-specific mecha-
nism, whereby a viral/plasmid integrase gene recombines into a
host tRNA gene and partitions (32). The capture of a genetic
element in a chromosome leaves a trace because the intN
fragment overlapping the tRNA gene is generally maintained,
even if the remainder of the genetic element degenerates or is
deleted (51, 52) (Table 2).
For strains REY15A and HVE10/4, remnants of integrated
elements adjoin eight and five tRNA genes, respectively
(Table 2). Most of the integrated genes derive from conju-
gative plasmids, and fuselloviral genes were detected only at
tRNAThr[GGT] in each strain, with an integrated region of
unknown origin at tRNAMet[CAT] in strain REY15A. All of
the integrated elements are highly degenerate, with IS elements
or miniature inverted-repeat transposable elements (MITEs) in-
serted downstream from the tRNA genes (Table 2). Given the
possibility of multiple integrations of genetic elements occurring
at a given tRNA gene, it is difficult to analyze unambiguously the
origins of residual integrated genes (42).
In contrast to the two Icelandic strains, the other S. solfa-
taricus and S. islandicus genomes carry intact genetic elements
bordered by intN and intC fragments that are all potentially
excisable (44, 52). They each show evidence of 2 to 7 tRNA
gene integration events, in which the most conserved sites are
tRNAPro[GGG] and tRNAAla[GGC], with less common events
at tRNALeu[GAG] and different alleles of tRNAArg(Table 2).
For the integrated tRNA genes of the Icelandic strains, there
was no significant correlation between the identity of the
tRNA anticodon and the frequency of codon usage or between
the encoded amino acid and the average number of amino
acids in the genome-encoded proteins.
Anti-integration role for tRNA introns. Each genome carries
45 tRNA genes and 2 to 3 pseudo-tRNA genes all located in
conserved regions. Sixteen of the tRNA genes contain introns
immediately 3? to the anticodon, varying in size from 12 to 65
bp, and in contrast to many archaeal tRNA genes, none were
detected at other sites (29), although putatively degenerate
introns, lacking the capacity to form splicing sites, occur in
D-loop regions of tRNAGlu[CTC] and tRNAGlu[TTC]. More-
over, the tRNA genes and introns are highly conserved in
sequence between the two genomes, and also with the other six
S. islandicus genomes, with very few base changes occurring
between the introns of a given tRNA. This high level of tRNA
and intron sequence conservation extends to S. solfataricus P2,
with only very minor differences observed for about one-third
of the genes, and it reinforces the concept that the RNA
introns are functionally important (5).
A possible function for the tRNA introns, suggested by
the above-described analyses, is that they provide protection
against integration of genetic elements into tRNA genes.
Integration can be disadvantageous in that pre-tRNA tran-
scription can be impaired. Only two intron-carrying tRNA
genes showed evidence of integration events (Table 2). For
the tRNAMet[CAT] gene copies, an intact integrase gene is
located downstream from the tRNA gene, while for the
tRNAPro[GGG], an overlapping intN fragment is present,
but the overlapping sequence does not extend to the intron,
suggesting that the intron entered after the integration
event. This is consistent with the latter integration event
being the most conserved, and probably the most ancient,
among Sulfolobus species.
IS elements and the versatile orfB element. Each genome
carries a limited range of IS element types, with some in mul-
tiple copies (Table 3). The IS elements are clustered in the
variable genomic region and also downstream from tRNA
genes that have undergone integration events (Fig. 1A). Many
of these elements appear to be intact, carrying the inverted
terminal repeats (ITRs) required for transposition, but exhibit
fragmented transposase genes, which are unlikely to be re-
stored by programmed translational frameshifting, as was ob-
served for some bacterial transposases of the IS1 and IS3
TABLE 2. Integration events at tRNA genes showing the sizes and origins of the residual integrated genes
SiRe1242-1247 conj plasmid
SiRe1321-1323 conj plasmid
SiRe1465-1479, 12 kb ? IS, pNOB8 integrase, unknown
SiRe1484-1490 conj plasmid
SiRe2413-2417 IS/MITEs SSV
SiRe1787-1792, 7 kb ? IS
SiH1399-1402 conj plasmid
SiH1561-1574 conj plasmid
aSSV, spindle-shaped fusellovirus; conj, conjugative; int, integrase.
VOL. 193, 2011GENOME ANALYSES OF ICELANDIC STRAINS OF S. ISLANDICUS1675
families (28). Although some of these elements may be mobi-
lizable by transposases acting in trans, for over one-third of the
IS families present, there is no encoded transposase (Table 3).
Potentially, the most active elements are ISC1200 and ISC1234
in both genomes and ISC1229 in strain HVE10/4 (Table 3).
The two Icelandic S. islandicus strains, together with those
from Kamchatka, Russia, carry the lowest number of IS ele-
ments (Table 1), many of which are inactive.
orfB elements of family IS605, together with elements of the
IS6 family (Table 3), are considered to represent the few
classes of transposable elements that are ancestral to the ar-
chaeal domain (16). orfB occurs alone, or together with a
transposase gene, orfA, in the IS200/605 family of transposable
elements. They lack ITRs, and both element types occur
commonly in viruses and conjugative plasmids of the Sulfolo-
bales (18, 40) (Table 3). Exceptionally, strain REY15A and
HVE10/4 genomes carry 11 and 16 nearly identical copies of
the single orfB elements in unconserved genomic positions,
respectively. This is consistent with these being the most active
transposable elements in each genome (Table 3), although it
remains uncertain whether they are autonomous or require an
OrfA in trans for mobility (16). In addition, the orfB elements
are exceptionally adaptable, because a further 8 and 2 copies
are physically coupled to copies of ISC1200 for strains
REY15A and HVE10/4, respectively (Table 3), and are poten-
Sulfolobus MITEs. Only two MITE types were detected in
multiple copies in each genome, SMN1 (320 bp) and SM3A
(164 bp) (Table 3), and both of which are capable of non-
autonomous transposition in different S. islandicus strains, fa-
cilitated by transposases of ISC1733 and ISC1058, respectively
(2, 4, 43). All SMN1 copies are located immediately down-
stream from the sequence TTTAA, but none occur at con-
served positions within the two genomes. Clearly, the SMN1
MITEs are active in both of the genomes, as is ISC1733, which
encodes the mobilizing transposase (Table 3), and they appear
to be cleanly excised when mobilized, in agreement with the
results of an earlier induced excision in the S. islandicus strain
REN1H1 (2). Although most SMN1 copies lie in intergenic
regions, and may or may not affect regulatory signals, some
appear to inactivate or alter genes. Thus, in strain REY15A,
an AAA?ATPase (SiRe0883) and a hypothetical gene
(SiRe0925) have incurred insertions in their promoters, and in
strain HVE10/4, SMN1 copies partially overlap with two genes
(SiH0773/2472), generating altered ORF sequences.
In contrast, the two SM3A copies are conserved in position
in each genome, consistent with the mobilizing transposase
encoded in ISC1058 being degenerate in both genomes. Nev-
ertheless, each SM3A copy retains the conserved 8-bp inverted
terminal repeat of the ISC1058 element (and unconserved
9-bp direct repeats resulting from the transposition event) and
can potentially be mobilized if a transposase-encoding
ISC1058 element enters the cell. Their maintenance as intact
elements may result from one SM3A copy overlapping with the
start of a conserved C/D box RNA gene (3), which may alter its
transcriptional properties, while the other lies between pro-
moters of two conserved protein genes and may influence their
relative transcriptional levels. SM3A occurs in a few copies in
each of the sequenced S. islandicus genomes, whereas SMN1 is
limited to the Icelandic and three Kamchatka strains, where it
occurs in 1 to 5 copies (Table 1).
Strain-specific metabolic pathways. Each Icelandic strain
shows a few specific metabolic properties. Thus, the REY15/A
strain carries an operon (SiRe0441-0445) encoding enzymes
implicated in nitrate reduction and nitrite extrusion, suggesting
that it can use nitrate as a terminal electron acceptor for
anaerobic respiration. The operon is located in the variable
region and has been observed previously only for two other
TABLE 3. Properties of the IS elements, transposases, and MITEs in the Icelandic genomesa
aThe nomenclature used for IS elements and MITEs follows that which was used previously (2, 6, 16). For the OrfB elements, the numbers in parentheses indicate
the numbers of copies that are physically linked to ISC1200 elements. TPase, transposase.
1676GUO ET AL.J. BACTERIOL.
archaea, S. islandicus strains M.14.25 and M.16.27. The larger
genome of strain HVE10/4 exclusively carries a urease operon
(SiH0978-0983) predicted to encode enzymes involved in the
hydrolysis of urea to NH4and CO2and previously found only
in the archaea Sulfolobus tokodaii, Metallosphaera sedula, and
Cenarchaeum symbiosum. Moreover, uniquely for a Sulfolobus
species, strain HVE10/4 also carries several genes predicted to
encode hydrogenases and hydrogenase maturation enzymes
(SiH0883-0892) in the variable region, which suggests that the
strain may be able to grow anaerobically.
A 50-kb region of strain HVE10/4 in the variable region
(SiH0447-0489) is bordered by IS elements and carries 15
predicted glycosyl transferase genes (group 1 and family 2),
constituting about half of the genome copies, interspersed al-
most exclusively with genes of unknown function and a gene
encoding a predicted polysaccharide biosynthesis enzyme. It is
well established that Sulfolobus S-layer proteins SlaA and SlaB
(SiRe1612/1 and SiH1691/0, respectively) are heavily glycosyl-
ated (36), but the relatively low G?C content of the region
suggests that it has been inserted and has an alternative un-
known function. The genome region is absent from strain
REY15A and from some of the other S. islandicus strains
Transporters. Sulfolobus strains utilize different sugars and
carbohydrates as carbon and energy sources (19), consistent
with their coding capacity for solute ABC transporters. A total
of 15 different ABC transporters were identified, of which
strain REY15A carries 12 and strain HVE10/4 contains 14. Of
these, 11 ABC transporters are present in S. solfataricus P2
(53), 6 in S. tokodaii (23), but only 3 in S. acidocaldarius (9).
The other S. islandicus genomes each carry 10 to 14 ABC
transporters (44) (Table 1). In both of the Icelandic genomes,
many ABC transporter genes are located in the variable region
(Fig. 1A) and are often flanked by transposons, consistent with
their being subjected to loss or gain events.
The ABC transporters are diverse, and some of their solute
specificities have been identified for other Sulfolobus strains
(15, 24). Cellobiose, maltose, and arabinose transporters are
present in both of the Icelandic genomes and most other se-
quenced S. solfataricus and S. islandicus genomes, although a
few S. islandicus strains lack one of the systems, as follows: the
arabinose system is absent from strain YG5714, while the malt-
ose system is not present in strains YN1551 and LD215. Strik-
ingly, the transporter of glucose, the preferred carbon source
for many microbes, is present only in the Icelandic strains, S.
islandicus strains M1415 and YG5714, and in S. solfataricus P2.
The lack of specific ABC transporters suggests either that
glucose is an uncommon nutrient in hot environments or that
another ABC transporter can facilitate glucose transport. One
ABC transporter encoded in the variable region of strain
HVE10/4 (SiH0899-0903), flanked by IS elements, appears to
be unique in public sequence databases.
Toxin-antitoxin systems. Four of the eight families of anti-
toxin-toxin complexes characterized for free-living bacteria also
occur in archaea, of which the VapBC family is by far the most
abundant (34) and is the main antitoxin-toxin family that we
detected in the Sulfolobus strains. The Icelandic strains REY15A
and HVE10/4 carry 17 and 18 vapBC gene pairs, respectively
(Table 1), as well as 2 vapC-like gene copies coupled to other
genes. They are distributed throughout the genomes, with several
located in the variable region, and only five gene pairs are con-
served in sequence and gene contexts in both strains (SiRe0698/
building exercises demonstrated that the sequences of both
antitoxins and toxins within each genome are very diverse and can
be classified into subtypes (data not shown), consistent with their
functional diversity and targeting of different cellular sites. These
data also indicate, for given gene pairs, that the subtypes of VapB
and VapC do not always correspond, implying that some gene
pairs may have exchanged partners.
Reading frame shifts and mRNA intron splicing. Examples
of translational reading frame shifts yielding single polypep-
tides have been demonstrated experimentally for S. solfa-
taricus P2 (10). For two of these, a predicted transketolase
(SiRe1696/8 and SiH1776/8) and a putative O-sialoglycopro-
tein endopeptidase (SiRe1569/70 and SiH1648/9), the S. islan-
dicus genes overlap in a similar way and are likely to undergo
reading frame shifts. In contrast to S. solfataricus P2, ?-fuco-
sidase (SiRe2185 and SiH2241) is a single gene, as is the
predicted dihydrolipoamide acyltransferase gene (SiH0582),
located only in strain HVE10/4. Very few transposase genes
present in IS elements (Table 3) carry a single reading frame
shift that could be expressed as a single protein via transla-
tional reading frame shifts (28).
Transcripts of the intron-carrying cbf5 genes (SiRe1607/8
and SiH1686/7) have been demonstrated to be spliced by the
archaeal splicing enzyme at the mRNA level in some crenar-
chaea (60). Other mRNAs, including those encoding the XPD
helicase (SiRe1685/SiH1765), have been predicted to undergo
splicing, but experimental support is lacking (5).
Noncoding RNAs. Many untranslated RNAs have been
characterized for S. solfataricus and S. acidocaldarius using a
variety of techniques, including probing cell extracts for RNA
with K-turn binding motifs and generating cDNA libraries of
total cellular RNA extracts, as well as numerous antisense
RNAs (33, 55, 59, 61). Most of these RNAs were characterized
for nucleotide length and partial sequence, and several were
detected by more than one experimental approach. We have
reanalyzed all these different RNA entities and have annotated
the S. islandicus RNA homologs which are conserved in both
sequence and gene contexts. The total number of RNA genes
and their putative functions are given (Table 4).
As for other archaeal hyperthermophiles, each genome car-
ries many C/D box RNAs that methylate primarily rRNAs and
tRNAs (Table 4). In strains REY15A and HVE10/4, 18 and 16
TABLE 4. Summary of Sulfolobus conserved noncoding RNA genes
located in the two Icelandic genomes
No. of indicated RNAs in
genome of strain:
VOL. 193, 2011GENOME ANALYSES OF ICELANDIC STRAINS OF S. ISLANDICUS1677
C/D box RNAs target rRNAs, respectively, while 4 modify
tRNAs and a further 3 have unknown targets. Two copies of
H/ACA RNA genes are present in each genome which, together
with the aPus7 protein (SiRe1836 and SiH1908), generate pseu-
douridine-35 in pre-tRNATyrtranscripts (31). Each of these C/D
box and H/ACA box RNA genes can be detected in the other
available S. islandicus genomes, which underlines their functional
importance. Of these, only three RNA genes characterized for
other Sulfolobus strains, Sso-sR4, Sso-sR8, and Sso-92, were not
located in any S. islandicus genomes (33, 55). For the numerous
noncoding RNAs of unknown function, similar contents were
found for the two Icelandic strains (Table 4) and for the other S.
islandicus strains, with only a few variations (Table 1), thereby
underlining their functional importance.
Diversity of the CRISPR-based immune systems. The
CRISPR/Cas and Cmr modules all lie within the large variable
regions. They show marked heterogeneity in the number and
family (25, 48) and are unconserved in position between the
genomes (Fig. 1A). Whereas REY15A carries one paired
CRISPR/Cas module of the family I type and two family B
Cmr modules, HVE10/4 contains two paired CRISPR/Cas
modules of family I and III types and a single family B Cmr
module (48) (Fig. 3A and B). This diversity of CRISPR-based
systems also extends to the other S. solfataricus and S. islandi-
cus genomes (Table 1). Although the gene content and orga-
nization of the paired family I CRISPR/Cas modules are quite
conserved among crenarchaea (48), exceptionally, for strain
HVE10/4, the internal group of cas genes located between the
two leader regions is inverted (Fig. 3B), indicative of a rear-
rangement having occurred within the module, possibly via the
identical inverted repeat sequences of the bordering leader
regions (Fig. 3B).
The CRISPR loci of strain REY15A carry 115 and 93 spacer-
repeat units centered at position 733,000, while those of
HVE10/4 contain 116 and 101 repeat-spacer units and 35 and
14 repeat-spacer units centered at positions 364000 and
745000, respectively (Fig. 1A). No spacer sequence identity
was detected within, or between, the two Icelandic strains or
with the other S. solfataricus and S. islandicus genomes. None
of the available fully sequenced S. islandicus genomes (Table
1) have any spacers in common, in contrast to the S. solfatari-
cus strains P1, P2, and 98/2, which all share many identical
spacers (17, 25) despite their being as distant from one an-
other, phylogenetically, as the S. islandicus strains (Fig. 2).
Thus, it seems that diversification of genomic CRISPR loci can
occur either by simple spacer turnover or by horizontal transfer
of whole or partial CRISPR/cas cassettes. There is increasing
evidence for the latter mechanism being the most common one
in S. islandicus strains (17, 21).
Since many of the characterized viruses and plasmids of Sul-
folobus derive from Iceland, we analyzed the degree to which
CRISPR spacer sequences of the Icelandic strains yielded signif-
icant matches to genetic element sequences using an earlier ap-
proach examining nucleotide and translated sequences of the
spacers (25, 49). Several significant sequence matches were de-
tected for both of the genomes, primarily to rudiviruses, fusello-
viruses, and conjugative plasmids, all of which are abundant in
Icelandic hot springs (63), but also were detected in smaller num-
bers to other viruses and cryptic plasmids (Fig. 3B).
The genome analyses underline the potential importance of
S. islandicus strain REY15A as a model organism for molec-
FIG. 3. (A) Phylogenetic tree of Cmr2 and its homologues in all sequenced archaeal genomes generates 5 families, A to E. The two Icelandic strains
carry family B Cmr modules, for which the gene order is shown. Other sequenced S. islandicus and S. solfataricus strains also carry Cmr modules of family
B and, less frequently, families D or E, as indicated on the tree. (B) Schematic representations of the CRISPR/Cas cassettes in the two Icelandic strains,
from families III and I (orange and blue, respectively). Compositions of the individual CRISPR loci are shown, where each triangle represents a
spacer-repeat unit. Significant spacer matches to sequenced viruses and plasmids are color coded (red, rudiviruses; orange, lipothrixviruses; yellow,
fuselloviruses; green, bicaudaviruses; turquoise, turreted icosahedral viruses; blue, conjugative plasmids; and violet, cryptic plasmids).
1678 GUO ET AL.J. BACTERIOL.
ular genetic studies of the Sulfolobales, and crenarchaea in
general, for a variety of reasons. The genome size of 2.5 Mb is
minimal for a Sulfolobus species; moreover, the incidence of
mobile elements is relatively low (Table 1), and stable deletion
mutants can be readily isolated (14, 20). Furthermore, the high
incidence of diverse ABC transporter systems (Table 1) may
explain why S. islandicus (and S. solfataricus) is most commonly
isolated from enrichment cultures obtained from terrestrial
acidic hot springs, which is in contrast to, for example, S.
acidocaldarius, which carries only three ABC transporters (9,
The relatively high incidence of deletion mutants obtained
from strain REY15A occurs despite the presence of several
transposable elements. However, in both of the Icelandic
strains, many of the IS elements are degenerate or carry dis-
rupted transposase genes (Table 3), consistent with the “copy-
and-paste” transpositional mechanism of most classes of Sul-
folobus IS elements and their undetectably low reversibility
rate (4, 41). The inability to remove the elements by sponta-
neous deletion, which does occur in many bacteria (16), may
also explain the presence of antisense RNAs in Sulfolobus
species to regulate transposase activity (55). The Icelandic
strains do, however, carry many copies of orphan orfB ele-
ments and SMN1 MITEs, which are mobilized by a “cut-
and-paste” mechanism presumably through OrfA encoded
in IS element ISC1733 (2, 16). The SMN1 MITEs appear to
be specific to the Icelandic and Kamchatka strains (Table 1),
and they can generate genetic novelty, reversibly, by extend-
ing open reading frames, in contrast to the other Sulfolobus
MITEs, which carry many potential stop codons in all read-
ing frames (43). The absence of most of the known Sulfolo-
bus MITEs, except SM3A, probably reflects the much lower
diversity of the mobilizing transposases present (Table 3).
Many of these elements are located in the large variable
region where genetic diversification occurs, including the
uptake and loss of operons and gene cassettes and rear-
rangements of mainly nonessential genes. A similar variable
genetic region in many genetic elements of Sulfolobus has
also been observed (e.g., see reference 18).
Many questions concerning the exceptional molecular and
cellular properties of crenarchaeal organisms remain to be
resolved. They include the functions of the multiple and highly
diverse gene pairs encoding VapBC antitoxin-toxins. For hy-
perthermophilic Sulfolobus species, in particular, their pres-
ence and variety could be a prerequisite for adaptation to life
under extreme, and sometimes rapidly varying, temperature
and pH conditions, as well as to survival in nutrient-poor en-
vironments possibly by optimizing the quality control of gene
expression (12, 34). They may also be related to the sulfolo-
bicins implicated in killing competitor Sulfolobus cells (39).
The crystal structure of a VapC toxin from the crenarchaeal
hyperthermophile Pyrobaculum aerophilum implicated the pro-
tein in exonuclease activity (1), but the multiplicity and wide
sequence diversity of the vapBC genes suggest that the toxins
target different cellular or molecular sites.
Strain HVE10/4 has been used as a host for a variety of
genetic elements, mainly from Iceland, which were likely to be
genetically close to the Icelandic host (63). The genome anal-
yses provide few insights into why it is a good host, especially
since it appears to carry a type 1 restriction-modification sys-
tem (SiH1435 to SiH1437). Moreover, the CRISPR/Cas and
CRISPR/Cmr modules of strain HVE10/4 are relatively com-
plex, as they also are for strain REY15A and other Sulfolobus
strains. Their activities have also been demonstrated, at least
for strain REY15A, by challenging the CRISPR/Cas systems
with vector-borne matching protospacers maintained under
selection, which produced deletions of the matching spacers
(20). The puzzle remains as to why the Sulfolobus CRISPR-
based systems are so complex, given that many of the viruses
and plasmids coexist at low copy numbers and are nonlytic.
One possibility is that the CRISPR/Cmr system primarily has a
regulatory role, with antisense crRNAs (CRISPR RNAs) tar-
geting viral mRNAs. Whatever the reason, the genetic close-
ness of strains REY15A and HVE10/4 suggests that the former
may also be a broad host for viruses and plasmids, with the
added advantage that genetic manipulation systems are now
available, and our preliminary studies with fuselloviruses and
conjugative plasmids support this supposition.
This research was supported by grants from the National Natural
Science Foundation of China (grants 306210165, 30730003, and
30870058) to L.H., a grant from the Danish Research Council for
Technology and Production (grant 09-062932) to Q.S., and grants from
the Danish Natural Science Research Council (grant 272-08-0391) and
Danish National Research Foundation to R.A.G.
1. Arcus, V. L., K. Ba ¨ckbro, A. Roost, E. L. Daniel, and E. N. Baker. 2004.
Distant structural homology leads to the functional characterisation of an
archaeal PIN domain as an exonuclease. J. Biol. Chem. 279:16471–16478.
2. Berkner, S., and G. Lipps. 2007. An active nonautonomous mobile element
in Sulfolobus islandicus REN1H1. J. Bacteriol. 189:2145–2149.
3. Bize, A., et al. 2009. A unique virus release mechanism in archaea. Proc. Natl.
Acad. Sci. U. S. A. 106:11306–11311.
4. Blount, Z. D., and D. W. Grogan. 2005. New insertion sequences of Sulfolo-
bus: functional properties and implications for genome evolution in hyper-
thermophilic archaea. Mol. Microbiol. 55:312–325.
5. Bru ¨gger, K., X. Peng, and R. A. Garrett. 2007. Sulfolobus genomes: mecha-
nisms of rearrangement and charge, p. 95–104. In R. A. Garrett and H.-P.
Klenk (ed.), Archaea: evolution, physiology, and molecular biology. Black-
well Publishing, Oxford, United Kingdom.
6. Bru ¨gger, K., et al. 2002. Mobile elements in archaeal genomes. FEMS
Microbiol. Lett. 206:131–141.
7. Bru ¨gger, K., E. Torarinsson, P. Redder, L. Chen, and R. A. Garrett. 2004.
Shuffling of Sulfolobus genomes by autonomous and non-autonomous mo-
bile elements. Biochem. Soc. Trans. 32:179–183.
8. Brumfield, S. K., et al. 2009. Particle assembly and ultrastructural features
associated with the replication of the lytic archaeal virus Sulfolobus turreted
icosahedral virus. J. Virol. 83:5964–5970.
9. Chen, L., et al. 2005. The genome of Sulfolobus acidocaldarius, a model
organism of the Crenarchaeota. J. Bacteriol. 187:4992–4999.
10. Cobucci-Ponzano, B., et al. 2010. Functional characterisation and high-
throughput proteomic analysis of interrupted genes in the archaeon Sulfolo-
bus solfataricus. J. Proteome Res. 9:2496–2507.
11. Contursi, P., et al. 2006. Characterisation of the Sulfolobus host-SSV2 virus
interaction. Extremophiles 10:615–627.
12. Cooper, C. R., A. J. Daugherty, S. Tachdjian, P. H. Blum, and R. M. Kelly.
2009. Role of vapBC toxin-antitoxin loci in the thermal stress response of
Sulfolobus solfataricus. Biochem. Soc. Trans. 37:123–126.
13. Delcher, A. L., K. A. Bratke, E. C. Powers, and S. L. Salzberg. 2007. Iden-
tifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformat-
14. Deng, L., H. Zhu, Z. Chen, Y. X. Liang, and Q. She. 2009. Unmarked gene
deletion and host-vector system for the hyperthermophilic crenarchaeon
Sulfolobus islandicus. Extremophiles 13:735–746.
15. Elferink, M. G., S. V. Albers, W. N. Konings, and A. J. Driessen. 2001. Sugar
transport in Sulfolobus solfataricus is mediated by two families of binding
protein-dependent ABC transporters. Mol. Microbiol. 39:1494–1503.
16. File ´e, J., P. Siguier, and M. Chandler. 2007. Insertion sequence diversity in
archaea. Microbiol. Mol. Biol. Rev. 71:121–157.
17. Garrett, R. A., et al. 2011. CRISPR-based immune systems of the Sulfolo-
bales: complexity and diversity. Biochem. Soc. Trans. 39:51–57.
VOL. 193, 2011GENOME ANALYSES OF ICELANDIC STRAINS OF S. ISLANDICUS1679
18. Greve, B., S. Jensen, K. Bru ¨gger, W. Zillig, and R. A. Garrett. 2004. Genomic Download full-text
comparison of archaeal conjugative plasmids from Sulfolobus. Archaea
19. Grogan, D. W. 1989. Phenotypic characterization of the archaebacterial
genus Sulfolobus: comparison of five wild-type strains. J. Bacteriol. 171:6710–
20. Gudbergsdottir, S., et al. 2011. Dynamic properties of the Sulfolobus
CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne
viral and plasmid genes and protospacers. Mol. Microbiol. 79:35–49.
21. Held, N. L., A. Herrera, H. Cadillo-Quiroz, and R. J. Whitaker. 2010.
CRISPR associated diversity within a population of Sulfolobus islandicus.
PLoS One 5:e12988.
22. Jonuscheit, M., E. Martusewitsch, K. M. Stedman, and C. Schleper. 2003. A
reporter gene system for the hyperthermophilic archaeon Sulfolobus solfa-
taricus based on a selectable and integrative shuttle vector. Mol. Microbiol.
23. Kawarabayasi, Y., et al. 2001. Complete genome sequence of an aerobic
thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7. DNA Res.
24. Koning, S. M., S. V. Albers, W. N. Konings, and A. J. Driessen. 2002. Sugar
transport in (hyper)thermophilic archaea. Res. Microbiol. 153:61–67.
25. Lillestøl, R. K., et al. 2009. CRISPR families of the crenarchaeal genus
Sulfolobus: bidirectional transcription and dynamic properties. Mol. Micro-
26. Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved
detection of transfer RNA genes in genomic sequence. Nucleic Acids Res.
27. Lundgren, M., A. Andersson, L. Chen, P. Nilsson, and R. Bernander. 2004.
Three replication origins in Sulfolobus species: synchronous initiation of
chromosome replication and asynchronous termination. Proc. Natl. Acad.
Sci. U. S. A. 101:7046–7051.
28. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol.
Biol. Rev. 62:725–774.
29. Marck, C., and H. Grosjean. 2003. Identification of BHB splicing motifs in
iintron-containing tRNAs from 18 archaea: evolutionary implications. RNA
30. Martusewitsch, E., C. W. Sensen, and C. Schleper. 2000. High spontaneous
mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is
mediated by transposable elements. J. Bacteriol. 182:2574–2581.
31. Muller, S., et al. 2009. Deficiency of the tRNATyr:?35-synthase aPus7 in
Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-
guided machinery. Nucleic Acids Res. 37:1308–1322.
32. Muskhelishvili, G., P. Palm, and W. Zillig. 1993. SSV1-encoded site-specific
recombination system in Sulfolobus shibatae. Mol. Gen. Genet. 273:334–342.
33. Omer, A. D., M. Zago, A. Chang, and P. P. Dennis. 2006. Probing the
structure and function of an archaeal C/D-box methylation guide sRNA.
34. Pandey, D. P., and K. Gerdes. 2005. Toxin-antitoxin loci are highly abundant
in free-living but lost from host-associated prokaryotes. Nucleic Acids Res.
35. Peng, N., Q. Xia, Z. Chen, Y. X. Liang, and Q. She. 2009. An upstream
activation element exerting differential transcription activation on an
archaeal promoter. Mol. Microbiol. 74:928–939.
36. Peyfoon, E., et al. 2010. The S-layer glycoprotein of the crenarchaeote Sul-
folobus acidocaldarius is glycosylated at multiple sites with chitobiose-linked
N-glycans. Archaea pii:754101.
37. Prangishvili, D., et al. 1998. Conjugation in archaea: frequent occurrence of
conjugative plasmids in Sulfolobus. Plasmid 40:190–202.
38. Prangishvili, D., P. P. Forterre, and R. A. Garrett. 2006. Viruses of the
Archaea: a unifying view. Nat. Rev. Microbiol. 4:837–848.
39. Prangishvili, D., et al. 2000. Sulfolobicins, specific proteinaceous toxins pro-
duced by strains of the extremely thermophilic archaeal genus Sulfolobus. J.
40. Prangishvili, D., et al. 2006. Structural and genomic properties of the hy-
perthermophilic archaeal virus ATV with an extracellular stage of the re-
productive cycle. J. Mol. Biol. 359:1203–1216.
41. Redder, P., and R. A. Garrett. 2006. Mutations and rearrangements in the
genome of Sulfolobus solfataricus P2. J. Bacteriol. 188:4198–4206.
42. Redder, P., et al. 2009. Four newly isolated fuselloviruses from extreme
geothermal environments reveal unusual morphologies and a possible inter-
viral recombination mechanism. Environ. Microbiol. 11:2849–2862.
43. Redder, P., Q. She, and R. A. Garrett. 2001. Non-autonomous elements in
the crenarchaeon Sulfolobus solfataricus. J. Mol. Biol. 306:1–6.
44. Reno, M. L., N. L. Held, C. J. Fields, P. V. Burke, and R. J. Whitaker. 2009.
Sulfolobus islandicus pan-genome. Proc. Natl. Acad. Sci. U. S. A. 106:8605–
8610. (Erratum, 106:18873.)
45. Robinson, N. P., and S. D. Bell. 2007. Extrachromosomal element capture
and the evolution of multiple replication origins in archaeal chromosomes.
Proc. Natl. Acad. Sci. U. S. A. 104:5806–5811.
46. Robinson, N. P., et al. 2004. Identification of two origins of replication in the
single chromosome of the archaeon Sulfolobus solfataricus. Cell 116:25–38.
47. Rutherford, K., et al. 2000. Artemis: sequence visualization and annotation.
48. Shah, S. A., and R. A. Garrett. 2011. CRISPR/Cas and Cmr modules,
mobility and evolution of adaptive immune systems. Res. Microbiol. 162:
49. Shah, S. A., N. R. Hansen, and R. A. Garrett. 2009. Distributions of CRISPR
spacer matches in viruses and plasmids of crenarchaeal acidothermophiles
and implications for their inhibitory mechanism. Trans. Biochem. Soc. 37:
50. She, Q., et al. 2008. Host-vector systems for hyperthermophilic archaeon
Sulfolobus, p. 151–156. In S.-J. Liu and H. L. Drake (ed.), Microbes and the
environment: perspective and challenges. Science Press, Beijing, China.
51. She, Q., X. Peng, W. Zillig, and R. A. Garrett. 2001. Gene capture in archaeal
chromosomes. Nature 409:478.
52. She, Q., B. Shen, and L. Chen. 2004. Archaeal integrases and mechanisms of
gene capture. Biochem. Soc. Trans. 22:222–226.
53. She, Q., et al. 2001. The complete genome of the crenarchaeon Sulfolobus
solfataricus P2. Proc. Natl. Acad. Sci. U. S. A. 98:7835–7840.
54. She, Q., et al. 2009. Genetic analyses in the hyperthermophilic archaeon
Sulfolobus islandicus. Biochem. Soc. Trans. 37:92–96.
55. Tang, T.-H., et al. 2005. Identification of novel non-coding RNAs as poten-
tial antisense regulators in the archaeon Sulfolobus solfataricus. Mol. Micro-
56. Torarinsson, E., H.-P. Klenk, and R. A. Garrett. 2005. Divergent transcrip-
tional and translational signals in Archaea. Environ. Microbiol. 7:47–54.
57. Wagner, M., et al. 2009. Expanding and understanding the genetic toolbox of
the hyperthermophilic genus Sulfolobus. Biochem. Soc. Trans. 37:97–101.
58. Worthington, P., V. Hoang, F. Perez-Pomares, and P. Blum. 2003. Targeted
disruption of the alpha-amylase gene in the hyperthermophilic archaeon
Sulfolobus solfataricus. J. Bacteriol. 185:482–488.
59. Wurtzel, O., et al. 2010. A single-base resolution map of an archaeal tran-
scriptome. Genome Res. 20:133–141.
60. Yokobori, S., et al. 2009. Gain and loss of an intron in a protein-coding gene
in Archaea: the case of an archaeal RNA pseudouridine synthase gene. BMC
Evol. Biol. 9:198.
61. Zago, M. A., P. P. Dennis, and A. D. Omer. 2005. The expanding world of
small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus. Mol.
62. Zhang, C., et al. 2010. Revealing the essentiality of multiple archaeal pcna
genes using a mutant propagation assay based on an improved knockout
method. Microbiology 156:3386–3397.
63. Zillig, W., et al. 1998. Genetic elements in the extremely thermophilic
archaeon Sulfolobus. Extremophiles 2:131–140.
1680 GUO ET AL.J. BACTERIOL.