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

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2009, p Vol. 75, No /09/$ doi: /aem Copyright 2009, American Society for Microbiology. All Rights Reserved. Biodiversity of Thermophilic Prokaryotes with Hydrolytic Activities in Hot Springs of Uzon Caldera, Kamchatka (Russia) 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-Osmolovskaya 1 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia 1 ; Bioengineering Center, Russian Academy of Sciences, Moscow, Russia 2 ; Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia 3 ; and L Institut National de la Recherche Agronomique, Nantes, France 4 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 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. * Corresponding author. Mailing address: Prospekt 60-Letiya Oktyabrya 7/2, Moscow, Russia. Phone: Fax: kublanov.ilya@gmail.com. Supplemental material for this article may be found at Published ahead of print on 31 October 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], -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 morphology 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 twostep 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 286

2 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 Spring, a description, and coordinates Sery, ETF; growth of gray filaments around the margins; N, E Temp ( C) ph Substrate Designation of enrichment Growth b revealed by microscopy Agarose 1507ag Abundant growth of cocci Casein 1507cas Moderate growth of single cocci -Keratin 1507a-ker Moderate growth of motile rods, small irregular cocci Shumny, ETF 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 Agarose 1518ag Moderate growth of rods covered with fine multilayered Casein 1518cas Weak growth of rods and cocci deposits (black/white/reddish); N, E -keratin 1518a-ker Abundant growth of rods and cocci Vertoletny, ETF 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 growth on the surfaces of sediments; N, E Thermophilny, ETF; white filaments, cyanobacterial mats; N, E Maly, OTF; decayed plant material (leaves, grass); N, E 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 - 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 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). Further cultivation of enrichment cultures and consequent 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 -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 Agarose 1524ag Abundant growth of long and short rods Chitin 1532chi Abundant growth of cocci a ETF, East thermal field; CTF, Central thermal field; OTF, Orange thermal field. b Weak, cells ml 1 ; moderate, to cells ml 1 ; and abundant, cells ml 1. 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.

3 288 KUBLANOV ET AL. APPL. ENVIRON. MICROBIOL. 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. 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 (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, respectively. Isolate 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 and 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. 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 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 polysaccharides, including microcrystalline cellulose (25). In this work, representatives of the genus Dictyoglomus were found in the cellulose-developing enrichments, and newly isolated strain , 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 grown on CMC and microcrystalline cellulose at 70 C and ph 20 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 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 (ph 20 C 6.6) activity was obtained with N- Isolate designation Original enrichment TABLE 2. Thermophilic isolates with hydrolytic activity obtained from in situ enrichments Closest relative % 16S rrna identity Hydrolyzed substrate(s) 96.9 Microcrystalline cellulose, carboxymethyl cellulose 96.8 Agarose Keratin, casein, albumin, gelatin 97.6 Microcrystalline cellulose, carboxymethyl cellulose cmc Dictyoglomus thermophilum strain Rt46B.1 T ag Dictyoglomus thermophilum strain Rt46B.1 T cas Caldanaerobacter subterraneous strain SEBR 7858 T 1523vc 1523rope Caldanaerobacter subterraneous strain SEBR 7858 T a-ker Fervidococcus fontis strain Keratin

4 VOL. 75, 2009 THERMOPHILIC PROKARYOTES WITH HYDROLYTIC ACTIVITIES 289 Downloaded from FIG. 3. Electron micrographs of negatively stained (25) strains (a) and (b) and a thin section (25) of cells of strain (c). Bars, 1 m. on October 12, 2018 by guest benzyloxycarbonyl-l-alanyl-l-alanyl-l-p-nitrophenylalanyl-l-phenylalanine -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(NO 2 )F-APM in a 5% water solution of DMFA (N,N-dimethylformamide) was added to 920 l of 0.02 M MOPS (morpholinepropanesulfonic acid), ph 20 C 6.6 (chymotrypsin-like activity), or of 0.1 M Na-acetate, ph 20 C 4.0 (pepsin-like activity), with 5 mm CaCl 2. 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, absorbance was measured at 320 nm (ε 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. 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 , isolated from the same enrichment. Production of extracellular proteinases with keratinolytic activity was previously shown for several representatives of the Thermanaerobacter-Caldanaerobacter group

5 290 KUBLANOV ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. Proteolytic activities of in situ enrichment cultures from Uzon Caldera hot springs Enrichment PF (ph 8.5) Activity ( /min) with a : Z- F(NO 2 ) F-APM (p 6.6) Z- F(NO 2 ) F-APM (p 4.0) Molecular mass(es) (kda) 1507a-ker cas a-ker b-ker a-ker al a-ker cas a-ker b-ker , 150, a-ker al cas , 90, 70 a A, alanine; P, proline; F, phenylalanine; Z, N-benzyloxycarbonyl. (21, 27). Indeed, in the supernatant of strain 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 , 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 Ca 2. 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 EU for strains , , 1523vc, and , respectively. The 16S rrna gene partial sequences for bacterial and archaeal DGGE bands were deposited in GenBank under accession numbers EU to EU for bacterial DGGE bands 1521a-ker 3, 1523b-ker 3, 1523cel 3, 1507cas 3, 1523rope 3, 1523gel 3, and 1521cmc 3, respectively, and EU to EU 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. 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