Biodiversity of Thermophilic Prokaryotes with Hydrolytic Activities in Hot Springs of Uzon Caldera, Kamchatka (Russia)

Abstract

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 α- or β-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 Caldicellulosiruptor 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 enrichments 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 prokaryotes 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 cultured 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 primers 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 prokaryotes identified by their 16S rRNA sequences (20). Colonization 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 containing insoluble biopolymers, allowing free access to surrounding hydrothermal fluids.

In a September 2005 expedition to Uzon Caldera, Kamchatka 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 [bovine; Sigma], casein [bovine; Sigma], α-keratin [porcine hair obtained from SIFDDA Co., Plouvara, France], and β-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 morphology of cells depended both on the substrate and on the spring characteristics (Table 1).

TABLE 1.

Characteristics of thermal sites of Uzon Caldera selected for in situ enrichment and enrichment cultures obtained from these sites

Spring,a description, and coordinates	Temp (°C)	pH	Substrate	Designation of enrichment	Growthb revealed by microscopy
Sery, ETF; growth of gray filaments around the margins; 54°29′58"N, 160°00′50"E	75	6.5	Agarose	1507ag	Abundant growth of cocci
	Casein	1507cas	Moderate growth of single cocci
	α-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
			α-Keratin	1510a-ker	Abundant growth of curved rods
			β-Keratin	1510b-ker	Moderate growth of cocci and single rodsi
Burlyashchy, CTF; sediments covered with fine multilayered deposits (black/white/reddish); 54°29′98"N, 160°00′11"E	86	7.0	Agarose	1518ag	Moderate growth of rods
	Casein	1518cas	Weak growth of rods and cocci
	α-keratin	1518a-ker	Abundant growth of rods and cocci
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
			α-Keratin	1521a-ker	Abundant growth of rods and cocci
Zatsepin, ETF; abundant lichen-like growth on the surfaces of sediments; 54°29′57"N, 160°00′40"E	70	7.0	Agarose	1523ag	Moderate growth of cells in sheaths, balls of filaments
	Cellulose	1523cel	Abundant growth of short rods
	Chitin	1523chi	Abundant growth of thick rods, long filaments
			Casein	1523cas	Moderate growth of irregular cells
			Albumin	1523alb	Moderate growth of diverse rods
			α-keratin	1523a-ker	Moderate growth of thick rods
			β-Keratin	1523b-ker	Abundant growth of short and long rods
			Linen rope	1523rope	Abundant growth of short rods
Thermophilny, ETF; white filaments, cyanobacterial mats; 54°49′83"N, 160°01′40"E	68	6.0	Agarose	1524ag	Abundant growth of long and short rods
Maly, OTF; decayed plant material (leaves, grass); 54°30′27"N, 160°00′02"E	87	4.1	Chitin	1532chi	Abundant growth of cocci

^aETF, East thermal field; CTF, Central thermal field; OTF, Orange thermal field.

^bWeak, 5 × 10⁶ cells·ml⁻¹; moderate, 1 × 10⁷ to 5 × 10⁷ cells·ml⁻¹; and abundant, ≥5 × 10⁷ cells·ml⁻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 Crenarchaeota (see Table S1 in the supplemental material), was performed in order to obtain material for denaturing gradient gel 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 cultivated taxa: those developing on α- and β-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 supplemental material). DGGE with archaeal primers revealed the presence of noncultivated archaea in cellulose-degrading enrichments. Organisms present in cellulolytic enrichments 1521cmc and 1523rope represented a deep lineage in the Crenarchaeota phylum (“unknown Desulfurococcales”), to which many uncultured organisms from Yellowstone, Iceland, and Kamchatka hot springs were found to belong (10, 12, 18). The first cultivated organism of this group is “Fervidococcus fontis,” isolated from Treshchinny Spring, Uzon Caldera (18).

FIG. 1.

Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of bacterial components (represented 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 indicated in brackets. Methanosarcina barkeri strain DSM 800, taken as an outgroup, was used to root the tree.

FIG. 2.

Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of archaeal components (represented 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 indicated in brackets. Methanosarcina barkeri strain DSM 800, taken as an outgroup, was used to root the tree.

Further cultivation of enrichment cultures and consequent isolation of pure cultures were performed using a basal medium described elsewhere (24). Ten grams per liter of the same polymeric substrates was added. The pH of the medium, adjusted with anoxic HCl or NaOH, and the cultivation temperature were approximately the same as those in the sites where in situ enrichments proceeded. Selected enrichments were repeatedly serially diluted to extinction in the same growth medium, 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 (α-keratin, casein, and gelatin) and cellulose, respectively. Isolate 1507-2 possessed coccoid cells, grew on α-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 represented the genus Dictyoglomus.

TABLE 2.

Thermophilic isolates with hydrolytic activity obtained from in situ enrichments

Isolate designation	Original enrichment	Closest relative	% 16S rRNA identity	Hydrolyzed substrate(s)
1521-1	1521cmc	Dictyoglomus thermophilum strain Rt46B.1T	96.9	Microcrystalline cellulose, carboxymethyl cellulose
1507-9	1507ag	Dictyoglomus thermophilum strain Rt46B.1T	96.8	Agarose
1523-1	1523cas	Caldanaerobacter subterraneous strain SEBR 7858T	95.8	α-Keratin, casein, albumin, gelatin
1523vc	1523rope	Caldanaerobacter subterraneous strain SEBR 7858T	97.6	Microcrystalline cellulose, carboxymethyl cellulose
1507-2	1507a-ker	“Fervidococcus fontis” strain 940	99.0	α-Keratin

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 μm.

The activities of corresponding hydrolytic enzymes in enrichment and pure cultures grown on polymeric substrates were measured. Cells of microorganisms and insoluble medium components were collected by centrifugation for 10 min at 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 terrestrial hot springs of different geographic locations (2, 6, 19), including Kamchatka (26). In contrast to what was found for Caldicellulosiruptor species, representatives of Thermoanaerobacteraceae were not known to be able to grow on cellulose. Newly isolated Caldanaerobacter sp. strain 1523vc used cellulose as the substrate for growth, extending our knowledge of the phenotypic diversity in this family. However, cellulase activity detected in the supernatant of strain 1523vc was relatively low: 1 μm 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 polysaccharides, 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 abundantly on cellulose and CMC, producing extracellular cellulase. The rates of CMC hydrolysis produced by the supernatant of isolate 1521-1 grown on CMC and microcrystalline cellulose at 70°C and pH^20°C 8.0 were evaluated as 124 μm and 36 μm 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 enrichment culture 1523ag showed extracellular glycosidase activity (as determined by a qualitative assay) at 75°C and pH^20°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-succinyl-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-benzyloxycarbonyl-l-alanyl-l-alanyl-l-p-nitrophenylalanyl-l-phenylalanine γ-morpholinopropylamide (28), synthesized and characterized 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-dimethylformamide) was added to 920 μl of 0.02 M MOPS (morpholinepropanesulfonic acid), pH²⁰°^C 6.6 (chymotrypsin-like activity), 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 μl of a proteinase-containing sample. The solution was incubated for 5 min. During incubation, absorbance was measured at 320 nm (ɛ₃₂₀ = 900 M⁻¹ cm⁻¹). The control samples were the same reaction mixture but devoid of proteinase solution. Table 3 summarizes the proteolytic activities of the studied enrichment cultures.

TABLE 3.

Proteolytic activities of in situ enrichment cultures from Uzon Caldera hot springs

Enrichment	Activity (μΜ/min) witha:			Molecular mass(es) (kDa)
	ΑΑPF (pH 8.5)	Z-ΑΑF(NO2) F-APM (pΗ 6.6)	Z-ΑΑF(NO2) F-APM (pΗ 4.0)
1507a-ker	0.06	0.9	0.44
1507cas	0	1.48	0.94	∼50
1507a-ker	0	0.54	2.27
1510b-ker	0	0	2.05
1510a-ker	0	0.57	2.86
1510al	0	0.38	0
1518a-ker	0	1.1	0.67
1518cas	0	0.43	0.26
1521a-ker	0.05	1.875	1.34	∼50
1523b-ker	0.064	0.65	0	∼200, ∼150, ∼80
1523a-ker	0.16	4.66	0
1523al	0.068	7.16	0
1523cas	0.015	8.0	0	∼220, ∼90, ∼70

^aA, alanine; P, proline; F, phenylalanine; Z, N-benzyloxycarbonyl.

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 produced by Caldanaerobacter sp. strain 1523-1, isolated from the same enrichment. Production of extracellular proteinases with keratinolytic activity was previously shown for several representatives of the Thermanaerobacter-Caldanaerobacter group (21, 27). Indeed, in the supernatant of strain 1523-1 culture growing on keratin, we found a ∼220-kDa thermostable keratinase, showing broad pH (6.0 to 10.0) and temperature (30 to 80°C) ranges of activity, with an optimum at pH 7.0 and 66°C. Addition of sodium dodecyl sulfate (optimally 0.35 mM) caused a 10-fold increase of activity of keratinase from strain 1523-1, while calcium positively influenced on the stability of the enzyme: 10-fold higher activity after 15 min of treatment at 100°C in the presence of 5 mM of Ca²⁺.

The presence of proteinases with molecular masses around ∼50 kDa was detected in in situ enrichments 1507cas and 1523a-ker populated mainly by coccoid cells, presumably of archaea (Table 1). Production of proteinases was shown for several hyperthermophilic archaea of both kingdoms (3, 8, 23). However, the archaea detected in proteinolytic enrichments were not hyperthermophiles but rather extreme thermophiles, growing at 70°C, and were distantly related to the Thermofilum genus (1510b-ker 2) or belonged to the “Fervidococcus” group (1510b-ker 1 and 1507cas 1) (Fig. 2).

In summary, the in situ enrichment cultures obtained in the presence of different polymeric substrates from Uzon hot springs demonstrate the diversity of thermophilic prokaryotes with hydrolytic activity inhabiting these springs. The obtained evidence also revealed a competition for substrates between different phylogenetic groups of prokaryotes and indicated a possible ecological function for the widespread but (until now) uncultured organisms.

Nucleotide sequence accession numbers.

The 16S rRNA gene partial sequences for products obtained by PCR with bacterial primers were deposited in GenBank under accession numbers EU183114, EU240006, EU851048, and EU240007 for strains 1523-1, 1521-1, 1523vc, and 1507-9, respectively. The 16S rRNA gene partial sequences for bacterial and archaeal DGGE bands were deposited in GenBank under accession numbers EU183107 to EU183113 for bacterial DGGE bands 1521a-ker 3, 1523b-ker 3, 1523cel 3, 1507cas 3, 1523rope 3, 1523gel 3, and 1521cmc 3, respectively, and EU216029 to EU216037 for archaeal DGGE bands 1507cas 1, 1510b-ker 1, 1510b-ker 2, 1521cmc 1, 1523rope 1, 1521cmc 2, 1507cas 2, 1523rope 2, and 1507ag 1, respectively.