APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2006, p. 5077–5082
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 7
16S rRNA Phylogenetic Investigation of the Candidate
Thomas A. Auchtung,1Cristina D. Takacs-Vesbach,2and Colleen M. Cavanaugh1*
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138,1
and Department of Biology, University of New Mexico, Albuquerque, New Mexico 871312
Received 9 January 2006/Accepted 15 April 2006
The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was
investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on
previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes
were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific
Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that
Korarchaeota exhibit a high level of endemicity.
In 1994, 16S rRNA gene sequences PCR amplified from
Yellowstone National Park hot spring Obsidian Pool were
reported (3). These sequences were hypothesized to belong to
an ancient division, the “Korarchaeota,” that diverged from the
Archaea lineage before the separation of Crenarchaeota and
Euryarchaeota (2). Other deeply branching clades have since
been proposed, including the Nanoarchaeota and “Ancient Ar-
chaeal Group,” although phylogenetic support for any group
having a basal position is not strong (2, 4, 29). What little is
known about Korarchaeota comes from detection of their 16S
rRNA genes during environmental diversity surveys that used
“universal” Archaea primers. Nineteen such sequences which
were believed to be in the Korarchaeota clade have been pub-
lished (3, 12, 15, 17, 19, 22–24, 28, 31, 32). These genes were
PCR amplified from fluid, sediment, sulfide chimneys, or mi-
crobial mats of high-temperature hot springs or submarine
vents. Although attempts to grow Korarchaeota in pure culture
have not been successful, Korarchaeota cells of the pJP27 phylo-
type originally detected in Obsidian Pool have now been highly
enriched, and their (meta)genome is being sequenced (K. O.
Stetter, J. G. Elkins, M. Keller, and the JGI-DOE [http://www.jgi
To further assess their diversity and distribution, we de-
signed Korarchaeota-specific primers based on published se-
quences and used them to PCR amplify 16S rRNA genes from
deep-sea hydrothermal vents, various Yellowstone National
Park hydrothermal features, and a number of nonhydrother-
mal environments. PCR amplification products were cloned
and sequenced, and the phylogeny of these 16S rRNA genes,
as well as those previously named Korarchaeota or sharing
identity to those previously named as such, was inferred. The
relationships among Korarchaeota were compared to the prop-
erties of the environments from which they were amplified to
gain insights into basic physiological properties of this enig-
Diverse deep-sea hydrothermal vent niches were screened
for Korarchaeota. Samples, collected from the 9°N East Pacific
Rise (EPR) 2,500-m-deep hydrothermal field in December
2002 using DSV Alvin on R/V Atlantis cruise AT-07, Leg 26,
were from the following environments: the dark red surface of
a black smoker sulfide chimney through which 370°C fluid
flowed (P-vent; Dive 3851), a basalt settlement piece (FRIE
16.2) left by an active sulfide chimney for 1 week (Tica vent;
Dives 3843 and 3849), and a 0.45-?m-pore-size, 142-mm-diam-
eter mixed cellulose ester filter (Millipore) and a 1-?m-pore-size,
142-mm-diameter Petex prefilter (Sefar) through which 200 liters
of water 1 m peripheral to a hydrothermal vent was filtered by
using a McLane Research Laboratories, Inc., pump in situ
(Tica vent). On board ship, sulfide chimney surfaces were
scraped into 2-ml freezer tubes and dropped in liquid nitrogen,
basalt pieces were placed in 50-ml polypropylene tubes, and
filters were wrapped in aluminum foil. Samples were stored on
board at ?70°C, transported on dry ice, and then stored at
?80°C until use. To extract DNA from sulfide chimney sam-
ples, the Bio 101 FastDNA SPIN kit for soil was used accord-
ing to the manufacturer’s instructions. DNA was extracted
from basalt and filters (in 50-ml polypropylene tubes) by the
addition of sucrose-lysis buffer (10 ml), lysozyme, proteinase K,
sodium dodecyl sulfate, and phenol-chloroform according to
the method of Gordon and Giovannoni (11) and then ethanol
precipitated (20) and resuspended in water.
A number of hydrothermal features in Yellowstone National
Park were also screened for Korarchaeota. Samples were col-
lected from 19 sites in summer 2003 and 17 sites in summer
2004 as part of a microbial inventory being conducted of Yel-
lowstone thermal features. Sediment, sinter, mud, and micro-
bial mats were mixed 1:1 in sucrose-lysis buffer, and hydrother-
mal fluid was mixed 2:1 with RNAlater (Ambion) on site and
then stored at ?80°C as soon as possible. DNA was extracted
from sediment, sinter, and mud by using a Bio 101 FastDNA
SPIN kit for soil. For extracting microbial mat DNA, 50 ?l of
thawed sample was mixed with 100 ?l of CTAB buffer (1%
cetyltrimethylammonium bromide, 0.75 M NaCl, 50 mM Tris
[pH 8], 10 mM EDTA) and then processed like the deep-sea
hydrothermal vent basalt and filters. For fluid samples, 2-ml
portions were centrifuged 10 min at 16,000 ? g, all but 50 ?l of
* Corresponding author. Mailing address: Harvard University, Bio-
logical Laboratories 4083, 16 Divinity Ave., Cambridge, MA 02138.
Phone: (617) 495-2177. Fax: (617) 496-6933. E-mail: cavanaug@fas
supernatant was removed, and then the samples were pro-
cessed as described for microbial mat samples.
Since Korarchaeota-specific primers had not previously been
used to screen natural samples, a range of readily available,
primarily nonhydrothermal environmental samples were also
collected and tested for Korarchaeota 16S rRNA genes. These
included compost (with temperatures measured up to 60°C),
plant leaves, flower petals, tree bark, human saliva, biofilms on
computer mice, a mosquito, soil (5- to 10-cm-deep Harvard
Forest, Petersham, MA, and surface soil of Cambridge, MA),
sand (Harvard University campus), river water and sediment
(Charles River, Cambridge, MA), lake water (oxic-anoxic in-
terface of Lake Mishawum, Woburn, MA), seawater (coast of
Nahant, MA), 32°C spring water (Chena, AK), 57°C tap water
(Cambridge, MA), and 60 and 90°C steam exhaust biofilms
(campus of Massachusetts Institute of Technology, Cambridge,
MA). Except the DNA of sediment, soil, and sand that was
extracted by using a Bio 101 FastDNA SPIN kit for soil, DNA
was extracted as for Yellowstone microbial mat samples.
For primer design, fifteen sequences of variable size identi-
fied as belonging to the Korarchaeota (3, 12, 15, 17, 19, 22, 23,
31, 32) were manually aligned in BioEdit (http://www.mbio
.ncsu.edu/BioEdit/bioedit.html) with a number of Euryarcha-
eota, Crenarchaeota, and Nanoarchaeota sequences. Two
“Korarchaeota” sequences (FZ2bA214 and pOWA133 [22,
31]) that shared very little identity with the other 13 were
excluded for the design of the Korarchaeota-specific primers
Kor236F (GCT GAG GCC CCA GGR TGG GAC CG) and
Kor1236R (CAT CCC GCT GTC CCG CCC ATT GC; num-
bering corresponds to Escherichia coli positions). Kor236F
had a one-half mismatch (degenerate position) with the nine
Korarchaeota sequences containing those regions, while con-
taining at least five and a half mismatches with all of the other
sequences in GenBank (determined by using Ribosomal
Database Project-II release 8.1 program Probe Match ).
Kor1236R had no mismatches with the 11 Korarchaeota se-
quences containing those regions while containing at least
three mismatches with all other sequences in GenBank. For
every sample that was screened by PCR, Kor236F and Kor1236R
were used together and individually with Univ1492R (Archaea/
Bacteria universal primer ) or Arc26F (Archaea universal
primer, TCC GGT TGA TCC TGC CGG A), respectively.
Univ1492R has no mismatches to the sequenced ends of Korar-
chaeota 16S rRNA genes (unpublished data), and Arc26F has
unknown identity to Korarchaeota 16S rRNA genes, since the 5?
end of a Korarchaeota 16S rRNA gene has not yet been se-
Control reactions were performed to determine the optimal
annealing temperatures of the Korarchaeota 16S rRNA gene
screening PCR. These were the lowest temperatures at which
marine group I Crenarchaeota (TFA4) and marine group II
Euryarchaeota (TFA3) 16S rRNA gene negative controls did
not amplify enough DNA to be visualized on an ethidium
bromide-stained agarose gel. Clones TFA3 and TFA4 were
created from PCR products that were Arc26F-Univ1492R
PCR amplified from DNA of the deep-sea hydrothermal vent
water described above.
When we screened for Korarchaeota 16S rRNA genes, pos-
itive control reactions (45°C annealing temperature) with
universal Archaea (Arc26F-Univ1492R) and/or Bacteria
(Bac27F -Univ1492R) primers yielded at least one cor-
rectly sized band for all samples, suggesting that samples that
did not yield products in Korarchaeota-specific PCR did not fail
due to inhibition or lack of DNA. As additional positive con-
trols, KorY38 plasmid(describedbelow)wasaddedto,andDNA
was successfully amplified from, replicates of the Kor236F-
Kor1236R reaction of nonhydrothermal samples and a rep-
licate of each Korarchaeota-specific PCR for Yellowstone
mM deoxynucleoside triphosphates, 4 mM MgCl2, 200 nM con-
centrations of each primer, and 0.04% bovine serum albumin.
After an initial denaturation of 94°C for 2 min, there were 35
cycles of 94°C for 45 s, 55°C (Kor236F-Univ1492R) or 62°C
(Arc26F-Kor1236R and Kor236F-Kor1236R) for 45 s, and 72°C
for 2 min, followed by 72°C for 2 min. A portion of each Korar-
chaeota-specific PCR-amplified sample was visualized on a 1%
agarose gel. When a band of the appropriate size was observed,
DNA in the remaining reaction volumes was purified by using a
PCR purification kit (QIAGEN), cloned into pCR2.1 (Invitro-
gen) or pDrive (QIAGEN) and then transformed into TOP10 E.
coli (20). The resulting colonies were screened by PCR with the
primers M13F and M13R under the same conditions as for the
Kor236F-Univ1492R reactions to identify clones that contained
inserts of the appropriate size. At least one clone per reaction was
sequenced by using the primer M13F with an ABI BigDye ter-
minator v3.1 cycle sequencing kit on an ABI Prism 3100 genetic
analyzer. Initial classification of partial sequences (typically ?500
nt) was performed by using BLAST (1). If a Korarchaeota match
was also cloned, and at least one representative was sequenced in
an effort to obtain longer Korarchaeota 16S rRNA gene se-
quences. Full sequencing of the most complete Korarchaeota 16S
rRNA gene clone from every site was accomplished with M13R.
Only Korarchaeota partial sequences ?99% identical to others
found at a site were also fully sequenced, since we used a large
number of PCR cycles to detect low numbers of Korarchaeota, at
the expense of a low mutation rate that is optimal for studying
To screen for additional Korarchaeota genes in Korarcha-
eota-positive samples and for undetected Korarchaeota genes
in negative samples with correctly sized amplicons, additional
screening was conducted. First, another round of PCR and
cloning was performed on the original sample; however, the
PCR for this round was not as successful and generally pro-
duced fewer clones. When fewer or greater than 10 clones were
generated, they were screened by sequencing or colony hybrid-
izations, respectively. For the latter, PCR with the primers
M13F and M13R was performed on four random clones to
confirm that they contained inserts. All colonies were then
lysed, and their DNA was fixed to Hybond N? membranes
.html). The DIG High-Prime DNA labeling and detection kit
(Roche) was used as directed, with one 70°C and two 80°C
washes; labeled probe was created from DNA Kor236F-
Kor1236R PCR amplified from the KorY42A plasmid (de-
scribed below). The Korarchaeota clones Kor9NEPR, KorY03, and
KorY38 (described below) were used as positive controls, and
Euryarchaeota (TFA3) and Crenarchaeota (TFA4) 16S rRNA
gene clones were used as negative controls. Although Korarcha-
5078AUCHTUNG ET AL.APPL. ENVIRON. MICROBIOL.
eota could not be proven definitively absent from samples in
which their 16S rRNA genes were not amplified, the positive
controls, multiple primer combinations, high number of PCR
cycles, and second round of screening decreased the possi-
bility that Korarchaeota were undiscovered due to technical
All new and previously identified putative Korarchaeota 16S
rRNA gene sequences were analyzed with the RDPII v8.1
program Chimera Check (6). Two recently published Korar-
chaeota sequences, WB3D011 and OPPE037 (24), were found
to be chimeras and excluded. Phylogenetic analyses included
31 possible Korarchaeota sequences. Ten of these were identi-
fied in the present study. Nineteen were previously published
as Korarchaeota, including the fifteen available when the prim-
ers were designed. Two sequences, pUWA9 and 20a-1, were
identified in GenBank by using BLAST. pUWA9 was pub-
lished as a Thermoplasmata 16S rRNA gene (31), and 20a-1 is
an unpublished sequence obtained from Aegean Sea sediment
(accession no. AJ299148). Putative Korarchaeota sequences
were compiled and aligned in ARB (14), with 43 16S rRNA
gene sequences from representatives of major archaeal groups
included to test the monophyly of Korarchaeota 16S rRNA
genes. Escherichia coli (Bacteria) and Homo sapiens (Eukarya)
rRNA genes were used as outgroups. After further refinement
of the alignment in BioEdit considering the predicted second-
ary structure of small subunit rRNA (5), alignments of various
lengths (651 nt, 119 to 770, n ? 20; 738 nt, 237 to 975, n ? 22;
946 nt, 29 to 975, n ? 17; 1,048 nt, 342 to 1,390, n ? 15; and
1,361 nt, 29 to 1,390, n ? 12) were exported to PAUP (27).
Trees were constructed and bootstrap resampled (500 repli-
cates) with maximum parsimony by 500 heuristic random-ad-
dition sequence searches using the tree bisection reconnection
branch-swapping option and treating gaps as new states.
To infer the relationships among all sequences within the
Korarchaeota, the phylogram from the analysis of the 946-nt
alignment (for which the most complete and phylogenetically
representative Korarchaeota sequences were available) was im-
ported into ARB, where individual partial sequences were
added to the tree by the parsimony add option, without chang-
ing the tree topology. The ARB-determined topological place-
ment and length of added partial sequence branches agreed
with those in individual trees generated from alignments spe-
cific to each partial sequence.
Ten putative Korarchaeota 16S rRNA genes were amplified
from 9 of the 63 diverse samples screened, all from sites at
temperatures of ?55°C, with a single sequence identified from
DNA of the active sulfide chimney (Kor9NEPR) and nine
sequences identified from 8 of the 41 Yellowstone samples
examined (Fig. 1). No chimeras were identified among these
sequences. Indels and nucleotide changes are nonconsecutive
in all sequences, except in a six-base loop region of KorY41B’s
Helix 44 (ARB helix numbering) that contains two insertions
and three changes compared to its most closely related se-
quences. KorY38 (amplified with Arc26F and Univ1492R) also
contains two and one additional mismatches with primers
Kor236F and Kor1236R, respectively, and 20a-1 has one ad-
ditional mismatch with Kor236F. Therefore, additional Korar-
chaeota-specific primers may be warranted in the future.
Phylogenetic analyses indicated that the proposed Korarcha-
eota sequences pOWA133 (31), FZ2bA214 (22), and pCIRA-X
(28), though first published as such, fall outside the group as
originally defined (2). These sequences diverge greatly from oth-
ers within the two largest regions (9-nt stems) conserved among
all Korarchaeota 16S rRNA gene sequences: Helix 6, where only
4 of 9 nt matched, and Helix 11, where each shared ?1 of 9 nt.
The original analyses of pOWA133 and pCIRA-X using neighbor
joining had placed these sequences in the Korarchaeota with only
weak support, while FZ2bA214 was named a Korarchaeota be-
cause it clustered with pOWA133 (22). Indeed, Takai et al. ques-
tioned their korarchaeotal identification of pOWA133 and have
since considered it a separate group (30).
The remaining 28 sequences formed a coherent clade des-
ignated here as Korarchaeota with 100% bootstrap support in
all trees. We define these as Korarchaeota because of their
strong phylogenetic support as a group (including the se-
quences originally named Korarchaeota) and conservation of
the two signature sequences described above. Although sup-
port for the hypothesis of a basal evolutionary position for
Korarchaeota remains weak, when bootstrap support was at all
present (?50%), Korarchaeota branched sister to the clade of
Crenarchaeota and Euryarchaeota (946-nt tree), with only the
pOWA133/FZ2bA214 clade (946-nt tree) or Ancient Archaeal
Group sequences (651-, 946-, and 1,361-nt trees) ever branch-
ing deeper (Fig. 1).
Analysis of the 28 Korarchaeota 16S rRNA gene sequences
indicate five subgroups exist based on various degrees of phy-
logenetic support and geographic location (Fig. 1). The overall
topology of trees constructed using other length sequence
alignments agreed with the 946-nt tree, except the 738-nt tree
in which groups I and II branched with group III Korarchaeota,
and groups IV and V were not resolved.
Group I sequences were all identified from Iceland samples.
Sequence 01A-11 was placed outside the clade containing
other group I and II sequences. However, this sequence is
provisionally included in group I because it shares five (non-
consecutive) nucleotides exclusively found in other group I
sequences and no nucleotides in common with just group II
sequences. KorY01, -03, -42A, and -55, all of which were am-
plified from Yellowstone National Park samples, are closely
related to each other and other group II sequences (99.0 to
99.8% identity). The sequence ST89 was too short (440 nt) to
resolve whether it is group I or group II. Group III, also
comprised of sequences amplified from Yellowstone National
Park samples, included KorY38, -41A, -42B, and -50.5, which
are closely related to one other and to two other previously
described sequences (98.6 to 99.7% identity). KorY41B, also
amplified from a Yellowstone hot spring, was basal in group
III. Group IV includes sequences amplified from samples col-
lected in or near the Pacific Ocean. Four sequences, recovered
from Japanese hot springs and submarine vents, form a clade
with relatively strong support (bootstrap 90; n ? 3 for analysis).
Support for other sequences clustering as a group with the
Japanese sequences was strong when longer sequence lengths
were used in analyses (1,048 nt, n ? 6, bootstrap 85; 1,361 nt,
n ? 4, bootstrap 97). Since all of these sequences also contain
an AT in Helix 24 that is not present in other Korarchaeota,
they are provisionally placed together in group IV. Group V
currently contains only two sequences (bootstrap 84), ampli-
fied from shallow and deep-sea hydrothermal vent samples
from the Mediterranean Sea and Indian Ocean, respectively.
VOL. 72, 2006 KORARCHAEOTA SURVEY 5079
FIG. 1. Phylogram of Korarchaeota based on 16S rRNA gene sequences. The samples, sites, and chemical and physical characteristics of the environments from which they were PCR amplified, length of
the sequences, accession numbers, and references are also listed. Major phylogenetic groupings are bracketed on the right. The tree topology and bootstrap values were generated by parsimony analysis of the
Escherichia coli, Homo sapiens, and 46 diverse Archaea small subunit rRNA genes were used as outgroups (shown with branches shortened and Euryarchaeota and Crenarchaeota branches collapsed). The scale
bar ? 5% change in nucleotide sequence. Superscript letters: a, hot springs and mudpots are terrestrial; b, F ? freshwater, S ? seawater, M ? mix, N ? not reported; c, numbering is based on E. coli; d, depth
below seawater surface (where not noted ? surface); e, the Korarchaeota clone naming terminology is “Kor” ? Y (Yellowstone) ? inventory sample number; f, EPR ? East Pacific Rise, CIR ? Central Indian
Ridge; g, a dash means not reported; h, sulfide chimney separated cold ambient seawater and hot hydrothermal fluid; i, previously referred to as Korarchaeota.
5080AUCHTUNG ET AL.APPL. ENVIRON. MICROBIOL.
Korarchaeota, currently found only in high-temperature hy-
drothermal settings, appear to be influenced by geographic
isolation, visible even from analysis of their very conserved 16S
rRNA gene sequences. Some microbial populations inhabiting
distantly separated extreme environments have been shown to
exhibit high levels of endemism consistent with geographic
isolation (18, 25, 35). However, to discern possible biogeo-
graphic patterns among Korarchaeota, additional surveys for
their 16S rRNA genes and other markers will be needed to
provide higher resolution.
High-temperature hydrothermal settings (50 to 128°C), in-
cluding sulfide chimney walls and caps, fluid, mud, sediment,
and microbial mats, are still the only places Korarchaeota 16S
rRNA genes have been detected (Fig. 1). To date, Korarcha-
eota 16S rRNA genes have not been retrieved in microbial
diversity surveys of chemically, physically, and proximally sim-
ilar but predominantly nonhydrothermal habitats, such as sul-
fide-rich cold springs, hydrocarbon seeps, anaerobic methane
oxidation-produced carbonate chimneys, deep-seawater or sedi-
ment, subseafloor samples, or off-axis deep-sea hydrothermal
vents (see, for example, references 7, 9, 16, 21, 33, and 34, where
the primers should have amplified known Korarchaeota 16S
rRNA genes equally well). The apparent thermophilic preference
of Korarchaeota is supported by the high G?C content of their
rRNA (mean ? 65.3% G?C [standard deviation ? 1.2]), a trait
that has been shown to correlate with high optimal growth tem-
perature (8). In contrast, the sample type, water source (marine
and freshwater), and pH (2.8 to 10.0) of environments found to
contain Korarchaeota 16S rRNA genes have been variable (Fig.
1). As microbial communities are characterized in ever more
detail, including metagenomic studies of as-yet-uncultured Korar-
chaeota, the environmental parameters which determine Korar-
chaeota distribution will be elucidated.
Over a decade since their discovery, almost nothing had
been revealed about representatives of the Korarchaeota. In-
sights into their environmental distribution and phylogeny pro-
vide a foundation for continuing the hunt for these enigmatic
organisms, their optimal conditions for growth in culture, and
for future genetic and metabolic studies.
Nucleotide sequence accession numbers. The sequences re-
ported in the present study have been deposited in GenBank
under accession numbers DQ465907-20 and DQ470015.
We thank Anna-Louise Reysenbach for conception of the Yellow-
stone microbial inventory; Kendra Mitchell, Olan Jackson-Weaver,
Mike Bobb, Sara Caldwell, George Silva, Chuck Fisher, the crew of the
R/V Atlantis, and the Alvin group for assistance in collection of hy-
drothermal samples; Meredith Fisher, Evan Lau, Tara Harmer, and
Chris Luke for selected samples from cool environments; Lief Fenno
for collection and analysis of the Chena, Alaska, spring water; and
Irene Newton for help with the colony hybridizations.
This study was supported by an NIH Genetics Training Grant grad-
uate fellowship to T.A.A., grants from NASA (NAG5-10906) and
NOAA National Undersea Research Center for the West Coast and
Polar Regions (03-0092) to C.M.C., and NSF Biotic Surveys and In-
ventories (0206773) to C.D.T.-V.
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