APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2009, p. 286–291 Vol. 75, No.

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0099-2240/09/$08.00⫹0 doi:10.1128/AEM.00607-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Biodiversity of Thermophilic Prokaryotes with Hydrolytic Activities in

Hot Springs of Uzon Caldera, Kamchatka (Russia)䌤†
Ilya V. Kublanov,1,4* Anna A. Perevalova,1 Galina B. Slobodkina,1 Aleksander V. Lebedinsky,1
Salima K. Bidzhieva,1 Tatyana V. Kolganova,2 Elena N. Kaliberda,3 Lev D. Rumsh,3
Thomas Haertlé,4 and Elizaveta A. Bonch-Osmolovskaya1
Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia1; Bioengineering Center, Russian Academy of
Sciences, Moscow, Russia2; Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Moscow, Russia3; and L’Institut National de la Recherche Agronomique, Nantes, France4
Received 13 March 2008/Accepted 22 October 2008

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87°C and pHs of 4.1
to 7.0, supplemented with proteinaceous (albumin, casein, or ␣- or ␤-keratin) or carbohydrate (cellulose,
carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated
at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several
enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel
electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR with Bacteria-specific
primers showed that the bacterial communities developing on carbohydrates included the genera Caldicellu-
losiruptor and Dictyoglomus and that those developing on proteins contained members of the Thermotogales
order. DGGE analysis performed after PCR with Archaea- and Crenarchaeota-specific primers showed that
archaea related to uncultured environmental clones, particularly those of the Crenarchaeota phylum, were
present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrich-
ments or corresponding natural samples of water and sediments represented the bacterial genera Dictyoglomus
and Caldanaerobacter as well as new archaea of the Crenarchaeota phylum. Thus, in situ enrichment and
consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial
communities of terrestrial hot springs.

Thermostable hydrolases produced by thermophilic pro-
karyotes are used in various industrial processes (4). However,
analyses of 16S rRNA genes in native DNAs from terrestrial
hot springs and deep-sea vents revealed the presence of many
thermophilic prokaryotes previously unknown and never cul-
tured in the laboratory and thus having virtually unknown
metabolic capacities (1, 7). A search for new thermostable
enzymes may also be performed by cloning genes directly from
bulk (metagenome) DNA isolated from hot springs (11). Its
success, however, depends greatly on the adequacy of the prim-
ers used.
Several attempts have previously been made to accumulate
the planktonic forms of thermophilic prokaryotes on surfaces
incubated in continuous contact with hydrothermal fluids. A
“vent cap” incubated in deep-sea hydrothermal fluid of the
Mid-Atlantic Ridge accumulated many new thermophilic pro-
karyotes identified by their 16S rRNA sequences (20). Colo-
nization by hyperthermophilic archaea of glass slide surfaces
during their incubation in New Zealand hot springs was also
reported (15). In this work, we tried to enrich thermophilic
microorganisms with hydrolytic activity trapped in tubes con-
taining insoluble biopolymers, allowing free access to sur-
rounding hydrothermal fluids.
* Corresponding author. Mailing address: Prospekt 60-Letiya Oktyabrya
7/2, 117312 Moscow, Russia. Phone: 74991354458. Fax: 74991356530. E-mail:
kublanov.ilya@gmail.com.
† Supplemental material for this article may be found at http://aem
.asm.org/.
䌤
Published ahead of print on 31 October 2008.
In a September 2005 expedition to Uzon Caldera, Kam-
chatka Peninsula, Russia, seven hot springs were selected for in
situ enrichment of thermophilic prokaryotes with hydrolytic
activities (Table 1). All springs were characterized by fairly
high water temperature (from 68 to 87°C) and neutral or
slightly acidic pH (4.1 to 7.0). Falcon tubes (15 ml) containing
200 to 300 mg of polymeric substrates (carboxymethyl cellulose
[CMC; Sigma], microcrystalline cellulose [Chemapol, Czech
Republic], chitin [crab chitin; Bioprogress, Russia], agarose
[agarose MP; Boehringer, Mannheim, Germany], albumin [bo-
vine; Sigma], casein [bovine; Sigma], ␣-keratin [porcine hair
obtained from SIFDDA Co., Plouvara, France], and ␤-keratin
[ground feathers]) were filled with thermal water, sealed with
screw caps, and placed in the spring studied. One-millimeter
perforations in the caps allowed exchange of fluid into and out
of the tube without loss of insoluble substrates precipitated at
the bottom of the tube. After 7 days of incubation, visible
degradation of polymeric substrates was observed in more than
half of the tubes, and the water covering the substrates turned
turbid. Light microscopy revealed abundant microbial growth
in the tubes with degraded substrates. The number and mor-
phology of cells depended both on the substrate and on the
spring characteristics (Table 1).
In the laboratory, DNA from several in situ enrichment
cultures was isolated as described previously (16), and a two-
step PCR with several sets of primers, universal and specific for
the domains Bacteria and Archaea and for the phylum Crenar-
chaeota (see Table S1 in the supplemental material), was per-
formed in order to obtain material for denaturing gradient gel

286
VOL. 75, 2009 THERMOPHILIC PROKARYOTES WITH HYDROLYTIC ACTIVITIES 287

TABLE 1. Characteristics of thermal sites of Uzon Caldera selected for in situ enrichment and enrichment cultures obtained from these sites
Temp Designation of
Spring,a description, and coordinates pH Substrate Growthb revealed by microscopy
(°C) enrichment

Sery, ETF; growth of gray filaments 75 6.5 Agarose 1507ag Abundant growth of cocci
around the margins; 54°29⬘58⬙N, Casein 1507cas Moderate growth of single cocci
160°00⬘50⬙E ␣-Keratin 1507a-ker Moderate growth of motile rods, small irregular cocci

Shumny, ETF 77 6.4 Agarose 1510ag Moderate growth of large cocci

Chitin 1510chi Moderate growth of diverse rods
Casein 1510cas Moderate growth of irregular cells
Albumin 1510alb Moderate growth of rods of diverse size
␣-Keratin 1510a-ker Abundant growth of curved rods
␤-Keratin 1510b-ker Moderate growth of cocci and single rodsi

Burlyashchy, CTF; sediments 86 7.0 Agarose 1518ag Moderate growth of rods

covered with fine multilayered Casein 1518cas Weak growth of rods and cocci
deposits (black/white/reddish); ␣-keratin 1518a-ker Abundant growth of rods and cocci
54°29⬘98⬙N, 160°00⬘11⬙E

Vertoletny, ETF 68 7.0 Cellulose 1521cmc Moderate growth of thick rods with rounded ends
and filaments
Chitin 1521chi Moderate growth of oval cells
Casein 1521cas Moderate growth of irregular cells
␣-Keratin 1521a-ker Abundant growth of rods and cocci

Zatsepin, ETF; abundant lichen-like 70 7.0 Agarose 1523ag Moderate growth of cells in sheaths, balls of
growth on the surfaces of filaments
sediments; 54°29⬘57⬙N, Cellulose 1523cel Abundant growth of short rods
160°00⬘40⬙E Chitin 1523chi Abundant growth of thick rods, long filaments
Casein 1523cas Moderate growth of irregular cells
Albumin 1523alb Moderate growth of diverse rods
␣-keratin 1523a-ker Moderate growth of thick rods
␤-Keratin 1523b-ker Abundant growth of short and long rods
Linen rope 1523rope Abundant growth of short rods

Thermophilny, ETF; white filaments, 68 6.0 Agarose 1524ag Abundant growth of long and short rods
cyanobacterial mats; 54°49⬘83⬙N,
160°01⬘40⬙E

Maly, OTF; decayed plant material 87 4.1 Chitin 1532chi Abundant growth of cocci
(leaves, grass); 54°30⬘27⬙N,
160°00⬘02⬙E
a
ETF, East thermal field; CTF, Central thermal field; OTF, Orange thermal field.
b
Weak, 5 ⫻ 106 cells 䡠 ml⫺1; moderate, 1 ⫻ 107 to 5 ⫻ 107 cells 䡠 ml⫺1; and abundant, ⱖ5 ⫻ 107 cells 䡠 ml⫺1.

electrophoresis (DGGE) assays. The corresponding methods

are described in detail elsewhere (18). DGGE analysis of 16S
rRNA genes present in field enrichment cultures showed a
diversity of bacteria and archaea (Fig. 1 and 2; also see Fig. S1
and Table S2 in the supplemental material). Most of bacteria,
detected in the in situ enrichment cultures, belonged to culti-
vated taxa: those developing on ␣- and ␤-keratins represented
the genus Fervidobacterium, and those growing on cellulose
and its derivatives represented the genera Dictyoglomus and
Caldicellulosiruptor (Fig. 1; also see Table S2 in the supple-
mental material). DGGE with archaeal primers revealed the
presence of noncultivated archaea in cellulose-degrading en-
richments. Organisms present in cellulolytic enrichments
1521cmc and 1523rope represented a deep lineage in the Cre- FIG. 1. Neighbor-joining tree based on 16S rRNA gene sequences
narchaeota phylum (“unknown Desulfurococcales”), to which showing the phylogenetic positions of bacterial components (repre-
many uncultured organisms from Yellowstone, Iceland, and sented by DGGE bands) of field enrichment cultures and related
Kamchatka hot springs were found to belong (10, 12, 18). The microorganisms. Bootstrap values (shown as percentages for 1,000
repetitions) are located at the branching points. The bar represents 10
first cultivated organism of this group is “Fervidococcus fontis,” substitutions per 100 nucleotide positions. GenBank numbers are in-
isolated from Treshchinny Spring, Uzon Caldera (18). dicated in brackets. Methanosarcina barkeri strain DSM 800, taken as
Further cultivation of enrichment cultures and consequent an outgroup, was used to root the tree.
288 KUBLANOV ET AL.
FIG. 2. Neighbor-joining tree based on 16S rRNA gene sequences
showing the phylogenetic positions of archaeal components (repre-
sented by DGGE bands) of field enrichment cultures and related
microorganisms. Bootstrap values (shown as percentages for 1,000
repetitions) are located at the branching points. The bar represents 10
substitutions per 100 nucleotide positions. GenBank numbers are in-
dicated in brackets. Methanosarcina barkeri strain DSM 800, taken as
an outgroup, was used to root the tree.
isolation of pure cultures were performed using a basal me-
dium described elsewhere (24). Ten grams per liter of the same
polymeric substrates was added. The pH of the medium, ad-
justed with anoxic HCl or NaOH, and the cultivation temper-
ature were approximately the same as those in the sites where
in situ enrichments proceeded. Selected enrichments were re-
peatedly serially diluted to extinction in the same growth me-
dium, and five isolates were obtained (Table 2). Isolates 1523-1
(Fig. 3a) and 1523vc, with short, rod-shaped cells, were found
to affiliate with the genus Caldanaerobacter (Table 2) and grew
on proteins (␣-keratin, casein, and gelatin) and cellulose, re-
spectively. Isolate 1507-2 possessed coccoid cells, grew on
␣-keratin or casein at 70°C and pH 6.0, and was found to be an
archaeon of the Crenarchaeota phylum, representing a cluster
of the so-called “unknown Desulfurococcales” (12, 18). Isolates
1507-9 and 1521-1 had filamentous cells, occasionally forming
clew-like structures (Fig. 3b and c). They grew at 70 and 80°C
and pH 6.5 on agarose and CMC, respectively, and repre-
sented the genus Dictyoglomus.
The activities of corresponding hydrolytic enzymes in enrich-
ment and pure cultures grown on polymeric substrates were
measured. Cells of microorganisms and insoluble medium
components were collected by centrifugation for 10 min at
TABLE 2. Thermophilic isolates with hydrolytic activity obtained from in situ enrichments
Isolate Original
Closest relative
designation enrichment
1521-1 1521cmc Dictyoglomus thermophilum strain
Rt46B.1T
1507-9 1507ag Dictyoglomus thermophilum strain
Rt46B.1T
1523-1 1523cas Caldanaerobacter subterraneous strain
SEBR 7858T
1523vc 1523rope Caldanaerobacter subterraneous strain
SEBR 7858T
1507-2 1507a-ker “Fervidococcus fontis” strain 940
APPL. ENVIRON. MICROBIOL.
10,000 rpm at 4°C, and hydrolytic activities in the resulting
supernatants were measured. The activities of glycosidases
were identified by measuring reduced sugar formation, using a
3.5-dinitrosalicilic reagent (13) with slight modifications.
Caldicellulosiruptor representatives detected in enrichment
cultures are known as active cellulolytics occurring in terres-
trial hot springs of different geographic locations (2, 6, 19),
including Kamchatka (26). In contrast to what was found for
Caldicellulosiruptor species, representatives of Thermoanaero-
bacteraceae were not known to be able to grow on cellulose.
Newly isolated Caldanaerobacter sp. strain 1523vc used cellu-
lose as the substrate for growth, extending our knowledge of
the phenotypic diversity in this family. However, cellulase ac-
tivity detected in the supernatant of strain 1523vc was relatively
low: 1 ␮m of reduced sugars produced per minute per ml of
sample. Dictyoglomus thermophilum, the type species of this
genus, was described as growing only on soluble substrates
(22), while Dictyoglomus turgidus, obtained previously from
Uzon Caldera, was found to grow weakly on solid polysaccha-
rides, including microcrystalline cellulose (25). In this work,
representatives of the genus Dictyoglomus were found in the
cellulose-developing enrichments, and newly isolated strain
1521-1, belonging to Dictyoglomus, was able to grow abun-
dantly on cellulose and CMC, producing extracellular cellu-
lase. The rates of CMC hydrolysis produced by the supernatant
of isolate 1521-1 grown on CMC and microcrystalline cellulose
at 70°C and pH20°C 8.0 were evaluated as 124 ␮m and 36 ␮m
of reduced sugars produced per minute per ml of the sample,
respectively.
Agarose was previously found to be hydrolyzed by a new
thermophilic bacterium, Caldanaerobacter uzonensis, isolated
from Thermophilny spring (I. Kozina, M. Hodges, K. Lee, I.
Wagner, J. Wiegel, I. Kublanov, and E. Bonch-Osmolovskaya,
submitted for publication), and the archaeon Desulfurococcus
fermentans (17). In this work, we found that high-melting-point
agarose was actively degraded in enrichments 1523ag and
1507ag by Dictyoglomus sp., easily identified by its specific
morphology. The supernatant of agarose-degrading enrich-
ment culture 1523ag showed extracellular glycosidase activity
(as determined by a qualitative assay) at 75°C and pH20°C.
The presence of proteinases and their molecular weights were
determined by a zymography method (9, 27). Peptidase activity
was determined using synthetic chromogenic substrate N-suc-
cinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide
(Suc-AAPF-pNa; Sigma Aldrich) as described in reference 9.
Chymotrypsin-like (pH20°C 6.6) activity was obtained with N-
% 16S rRNA
Hydrolyzed substrate(s)
identity
96.9 Microcrystalline cellulose, carboxymethyl cellulose
96.8 Agarose
95.8 ␣-Keratin, casein, albumin, gelatin
97.6 Microcrystalline cellulose, carboxymethyl cellulose
99.0 ␣-Keratin
VOL. 75, 2009 THERMOPHILIC PROKARYOTES WITH HYDROLYTIC ACTIVITIES 289

FIG. 3. Electron micrographs of negatively stained (25) strains 1523-1 (a) and 1507-9 (b) and a thin section (25) of cells of strain 1521-1 (c).
Bars, 1 ␮m.

benzyloxycarbonyl-L-alanyl-L-alanyl-L-p-nitrophenylalanyl-L-phe- proteinase solution. Table 3 summarizes the proteolytic activities

nylalanine ␥-morpholinopropylamide (28), synthesized and char-
acterized at the Shemyakin and Ovchinnikov Institute, Russian
Academy of Sciences. Sixty microliters of a 2.5 mM solution of
Z-AAF(NO2)F-APM in a 5% water solution of DMFA (N,N-di-
methylformamide) was added to 920 ␮l of 0.02 M MOPS (mor-
pholinepropanesulfonic acid), pH20°C 6.6 (chymotrypsin-like ac-
tivity), or of 0.1 M Na-acetate, pH20°C 4.0 (pepsin-like activity),
with 5 mM CaCl2. Upon stabilization of temperature, the reaction
was started by adding 20 ␮l of a proteinase-containing sample.
The solution was incubated for 5 min. During incubation, absor-
bance was measured at 320 nm (ε320 ⫽ 900 M⫺1 cm⫺1). The
control samples were the same reaction mixture but devoid of
of the studied enrichment cultures.
Bacteria of the genus Fervidobacterium are known to be able
to degrade proteins (5, 14). Keratinases of Fervidobacterium
species are membrane bound and consequently could not be
detectable, since only supernatants of in situ enrichments were
tested in this work. However, an extracellular enzyme with a
molecular mass of ⬃220 kDa and a neutral-to-alkaline pH
optimum, detected in enrichment 1523cas (Table 3), was pro-
duced by Caldanaerobacter sp. strain 1523-1, isolated from the
same enrichment. Production of extracellular proteinases with
keratinolytic activity was previously shown for several repre-
sentatives of the Thermanaerobacter-Caldanaerobacter group
290 KUBLANOV ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 3. Proteolytic activities of in situ enrichment cultures from 1521cmc 1, 1523rope 1, 1521cmc 2, 1507cas 2, 1523rope 2, and
Uzon Caldera hot springs 1507ag 1, respectively.
Activity (␮⌴/min) witha:
Molecular This work was supported by the Molecular and Cell Biology and
Enrichment Z-〈〈F(NO2) Z-〈〈F(NO2) mass(es)
〈〈PF Origin and Evolution of Biosphere programs of the Russian Academy
F-APM F-APM (kDa)
(pH 8.5) of Sciences, as well as by RFBR grant number 06-04-49045 and the
(p⌯ 6.6) (p⌯ 4.0)
Microbial Observatory in Kamchatka NSF grant.
1507a-ker 0.06 0.9 0.44
1507cas 0 1.48 0.94 ⬃50 REFERENCES
1507a-ker 0 0.54 2.27
1510b-ker 0 0 2.05 1. Barns, S., C. Delwiche, J. D. Palmer, and N. Pace. 1996. Perspectives on
archaeal diversity, thermophily and monophyly from environmental rRNA
1510a-ker 0 0.57 2.86
sequences. Proc. Natl. Acad. Sci. USA 93:9188–9193.
1510al 0 0.38 0 2. Bredholt, S., J. Sonne-Hansen, P. Nielsen, I. M. Mathrani, and B. K. Ahring.
1518a-ker 0 1.1 0.67 1999. Caldicellulosiruptor kristjanssonii sp. nov., a cellulolytic, extremely ther-
1518cas 0 0.43 0.26 mophilic, anaerobic bacterium. Int. J. Syst. Bacteriol. 49:991–996.
1521a-ker 0.05 1.875 1.34 ⬃50 3. Dib, R., J.-M. Chobert, M. Dalgalarrondo, G. Barbier, and T. Haertlé. 1998.
1523b-ker 0.064 0.65 0 ⬃200, ⬃150, Purification, molecular properties and specificity of a thermoactive and ther-
⬃80 mostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic
1523a-ker 0.16 4.66 0 archaea from deep-sea hydrothermal ecosystem. FEBS Lett. 431:279–284.
1523al 0.068 7.16 0 4. Egorova, K., and G. Antranikian. 2005. Industrial relevance of thermophilic
Archaea. Curr. Opin. Microbiol. 8:649–655.
1523cas 0.015 8.0 0 ⬃220, ⬃90, 5. Friedrich, A. B., and G. Antranikian. 1996. Keratin degradation by Fer-
⬃70 vidobacterium pennavorans, a novel thermophilic anaerobic species of the
a order Thermotogales. Appl. Environ. Microbiol. 62:2875–2882.
A, alanine; P, proline; F, phenylalanine; Z, N-benzyloxycarbonyl.
6. Huang, C. Y., B. K. Patel, R. A. Mah, and L. Baresi. 1998. Caldicellulosiruptor
owensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacte-
rium. Int. J. Syst. Bacteriol. 48:91–97.
(21, 27). Indeed, in the supernatant of strain 1523-1 culture 7. Hugenholtz, P., C. Pitulle, K. L. Hershberger, and N. R. Pace. 1998. Novel
division level bacterial diversity in a Yellowstone hot spring. J. Bacteriol.
growing on keratin, we found a ⬃220-kDa thermostable kera- 180:366–376.
tinase, showing broad pH (6.0 to 10.0) and temperature (30 to 8. Klingeberg, M., B. Galunsky, C. Sjoholm, V. Kasche, and G. Antranikian.
1995. Purification and properties of high thermostable, sodium dodecyl sul-
80°C) ranges of activity, with an optimum at pH 7.0 and 66°C. fate-resistant and stereospecific proteinase from extremely thermophilic ar-
Addition of sodium dodecyl sulfate (optimally 0.35 mM) chaeon Thermococcus stetteri. Appl. Environ. Microbiol. 61:3098–3104.
caused a 10-fold increase of activity of keratinase from strain 9. Kublanov, I. V., K. B. Tsiroulnikov, E. N. Kaliberda, L. D. Rumsh, T.
Haertle, and E. A. Bonch-Osmolovskaya. A keratinase from anaerobic ther-
1523-1, while calcium positively influenced on the stability of mophilic bacterium Thermoanaerobacter sp. strain 1004-09, isolated from a
the enzyme: 10-fold higher activity after 15 min of treatment at Baikal Lake rift zone. Mikrobiologiya, in press. (In Russian.)
100°C in the presence of 5 mM of Ca2⫹. 10. Kvist, T., B. K. Ahring, and P. Westermann. 2006. Archaeal diversity in
Icelandic hot springs. FEMS Microb. Ecol. 59:71–80.
The presence of proteinases with molecular masses around 11. Lian, M., S. Lin, and R. Zeng. 2007. Chitinase gene diversity at a deep sea
⬃50 kDa was detected in in situ enrichments 1507cas and station of the Pacific nodule province. Extremophiles 11:463–467.
12. Meyer-Dombard, D. R., E. L. Shock, and J. P. Amend. 2005. Archaeal and
1523a-ker populated mainly by coccoid cells, presumably of bacterial communities in geochemically diverse hot springs of Yellowstone
archaea (Table 1). Production of proteinases was shown for National Park, USA. Geobiology 3:211–227.
several hyperthermophilic archaea of both kingdoms (3, 8, 23). 13. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of
reducing sugar. Anal. Chem. 31:426–428.
However, the archaea detected in proteinolytic enrichments 14. Nam, G., D. Lee, H. Lee, N. Lee, B. Kim, E. Choe, J. Hwang, M Suhartono,
were not hyperthermophiles but rather extreme thermophiles, and Y. Pyun. 2002. Native-feather degradation by Fervidobacterium islandi-
growing at 70°C, and were distantly related to the Thermofilum cum AW-1, a newly keratinase-producing thermophilic anaerobe. Arch. Mi-
crobiol. 178:538–547.
genus (1510b-ker 2) or belonged to the “Fervidococcus” group 15. Niederberger, T. D., R. S. Ronimus, and H. W. Morgan. 2008. The microbial
(1510b-ker 1 and 1507cas 1) (Fig. 2). ecology of a high-temperature near neutral spring situated in Rotorua, New
In summary, the in situ enrichment cultures obtained in the Zealand. Microbiol. Res. 163:594–603.
16. Park, D. 2007. Genomic DNA isolation from different biological materials, p.
presence of different polymeric substrates from Uzon hot 3–13. In E. Hilario and J. Mackay (ed.), Protocols for nucleic acid analysis by
springs demonstrate the diversity of thermophilic prokaryotes nonradioactive probes, 2nd ed. Methods in molecular biology, vol. 353.
Humana Press, Inc., Totowa, NJ.
with hydrolytic activity inhabiting these springs. The obtained 17. Perevalova, A. A., V. A. Svetlichny, I. V. Kublanov, N. A. Chernyh, N. A.
evidence also revealed a competition for substrates between Kostrikina, T. P. Turova, B. B. Kuznetsov, and E. A. Bonch-Osmolovskaya.
different phylogenetic groups of prokaryotes and indicated a 2005. Desulfurococcus fermentans sp. nov., a novel hyperthermophilic
archaeon from a Kamchatka hot spring, and emended description of the
possible ecological function for the widespread but (until now) genus Desulfurococcus. Int. J. Syst. Evol. Microbiol. 55:995–999.
uncultured organisms. 18. Perevalova, A. A., T. V. Kolganova, N.-K. Birkeland, C. Schleper, E. A.
Nucleotide sequence accession numbers. The 16S rRNA Bonch-Osmolovskaya, and A. V. Lebedinsky. 2008. Distribution of Crenar-
chaeota representatives in terrestrial hot springs of Russia and Iceland. Appl.
gene partial sequences for products obtained by PCR with bac- Environ. Microbiol. 74:7620–7628.
terial primers were deposited in GenBank under accession num- 19. Rainey, F. A., A. M. Donnison, P. H. Janssen, D. Saul, A. Rodrigo, P. L.
bers EU183114, EU240006, EU851048, and EU240007 for Bergquist, R. M. Daniel, E. Stackebrandt, and H. W. Morgan. 1994. De-
scription of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: An obli-
strains 1523-1, 1521-1, 1523vc, and 1507-9, respectively. The 16S gately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Mi-
rRNA gene partial sequences for bacterial and archaeal DGGE crobiol. Lett. 120:263–266.
20. Reysenbach, A.-L., K. Longnecker, and J. Kirshtein. 2000. Novel bacterial
bands were deposited in GenBank under accession numbers and archaeal lineages from an in situ growth chamber deployed at a Mid-
EU183107 to EU183113 for bacterial DGGE bands 1521a-ker 3, Atlantic Ridge hydrothermal vent. Appl. Environ. Microbiol. 66:3798–3806.
1523b-ker 3, 1523cel 3, 1507cas 3, 1523rope 3, 1523gel 3, and 21. Riessen, S., and G. Antranikian. 2001. Isolation of Thermoanaerobacter
keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with ker-
1521cmc 3, respectively, and EU216029 to EU216037 for ar- atinolytic activity. Extremophiles 5:399–408.
chaeal DGGE bands 1507cas 1, 1510b-ker 1, 1510b-ker 2, 22. Saiki, T., Y. Kobayashi, K. Kawagoe, and T. Beppu. 1985. Dictyoglomus
VOL. 75, 2009 THERMOPHILIC PROKARYOTES WITH HYDROLYTIC ACTIVITIES
thermophilum gen. nov., sp. nov. a chemoorganotrophic, anaerobic, thermo-
philic bacterium. Int. J. Syst. Bacteriol. 35:253–259.
23. Sako, Y., P. C. Croocker, and Y. Ishida. 1997. An extremely heat-stable
extracellular proteinase (aeropyrolysin) from the hyperthermophilic
archaeon Aeropyrum pernix K1. FEBS Lett. 415:329–334.
24. Sokolova, T. G., N. A. Kostrikina, N. A. Chernyh, T. P. Tourova, T. V.
Kolganova, and E. A. Bonch-Osmolovskaya. 2002. Carboxydocella therm-
autotrophica gen. nov., sp. nov., a novel anaerobic, CO-utilizing thermo-
phile from a Kamchatkan hot spring. Int. J. Syst. Evol. Microbiol. 52:
1961–1967.
25. Svetlichny, V. A., and T. P. Svetlichnaya. 1988. Dictyoglomus turgidus sp.
nov., a new extremely thermophilic eubacterium isolated from hot springs of
the Uzon volcano caldera. Mikrobiologiya 57:364–369.
291
26. Svetlichny, V. A., T. P. Svetlichnaya, N. A. Chernykh, and G. A. Zavarzin.
1990. Anaerocellum thermophilum gen. nov., sp. nov., an extreme thermo-
philic cellulolytic eubacterium isolated from hot springs in the valley of
Geysers. Mikrobiologiya 59:598–604. (In Russian.)
27. Tsiroulnikov, K., H. Rezai, E. Bonch-Osmolovskaya, P. Nedkov, A. Goust-
erova, V. Cueff, A. Godfroy, G. Barbier, F. Métro, J.-M. Chobert, P. Clayette,
D. Dormont, J. Grosclaude, and T. Haertlé. 2004. Hydrolysis of the amyloid
prion protein and nonpathogenic meat and bone meal by anaerobic thermo-
philic prokaryotes and Streptomyces subspecies. J. Agric. Food Chem. 52:
6353–6360.
28. Zinchenko, A. A., L. D. Rumsh, and V. K. Antonov. 1977. Kinetic and
thermodynamic analysis of pepsin specificity. Sov. J. Bioorg. Chem. 3:1224–
1231.