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1 Unité de Microbiologie, INRA, Centre de Recherches de Clermont Ferrand – Theix, 63 122 Saint Genès – Champanelle, France
2 Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA
Correspondence
Annick Bernalier-Donadille
bernal{at}clermont.inra.fr
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CRE21T is AJ583243.
An unrooted neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships between strain CRE21T and some related members of the genus Bacteroides, a figure showing the kinetics of cellulose degradation by strain CRE21T and a table detailing the end products of cellulose fermentation by strain CRE21T are available as supplementary material with the online version of this paper.
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). Until recently, however, only a few Bacteroides species isolated from the human gut had been described (Salyers, 1984), most belonging to one of the ten species of the Bacteroides fragilis group. Recent investigations of this Bacteroides population have improved our knowledge of its structure, through isolation and description of novel species such as Bacteroides plebeius, Bacteroides coprocola, Bacteroides helcogenes, Bacteroides intestinalis, Bacteroides finegoldii and Bacteroides dorei (Kitahara et al., 2005; Bakir et al., 2006a, b, c). As Bacteroides species are numerically important, they are assumed to play key roles in the degradation and fermentation of organic matter present in the colon (Salyers, 1995). Some Bacteroides species were indeed shown to be able to obtain carbon and energy from hydrolysis of soluble or hydrated polysaccharides (Bétian et al., 1977; Salyers, 1995). In addition, degradation of maize bran, oat bran and wheat bran was evidenced in one strain of Bacteroides ovatus (Martin et al., 1998) while one Bacteroides sp. isolate, but which was not further identified, was reportedly able to use pure cellulose (Bétian et al., 1977). Although insoluble polysaccharides, mainly found in the plant cell wall (cereals, fruits, vegetables), constitute one of the main sources of carbohydrates that are extensively degraded by the human gut microbiota, the fibrolytic population involved in the breakdown of these substrates remains rather poorly explored. During the course of a study on the predominant cellulose-degrading community from the human colon, a Gram-negative bacterium that showed high 16S rRNA gene sequence similarity to members of the genus Bacteroides was isolated from a faecal sample. The present study describes this new isolate, designated strain CRE21T, and further proposes that it represents a novel species of the genus Bacteroides.
Strain CRE21T was isolated from faeces of a non-methane-excreting healthy volunteer (50 years old, male) by using a strictly anaerobic technique (Hungate, 1969). All liquid and solid media were prepared, dispensed and inoculated under 100 % O2-free CO2 gas. Freshly voided faeces (1 g) were transferred into 10 ml sterile anoxic mineral solution and serial 10-fold dilutions down to 10–11 were then carried out in mineral solution. These faecal dilutions were inoculated (0.3 ml) into liquid basal cellulolytic (BC) medium (10 ml per tube) (Robert & Bernalier-Donadille, 2003) with cell-wall spinach (CWS) fraction at a final concentration of 0.7 % as the sole energy source. CWS residue was obtained as described by Mosoni et al. (1993). After 15 days incubation at 37 °C, the presence of cellulolytic organisms in faecal dilution cultures was estimated by measuring carboxymethylcellulase (CMCase) activity. Detection of CMCase activity was performed by using an agar plate assay as described by Forano et al. (1994).
Strain CRE21T was isolated from the highest dilution faecal cultures showing CMCase activity (10–8). Isolation was carried out by using solid BC medium containing CWS as energy source and the roll-tube technique. After three to five successive subcultures on roll-tubes and broth BC medium, the isolate was examined for purity using phase-contrast microscopy, in CWS- and glucose- (2 g l–1) grown cultures. The medium used for further routine cultivation of strain CRE21T was BC medium while growth and nutrition studies of strain CRE21T were carried out in semi-synthetic BC medium (Robert et al., 2001). Cell morphology and motility were studied in 18-h glucose-grown cultures using phase-contrast microscopy and electron microscopy after negative staining of the whole-cell fraction with 2 % uranyl acetate. Gram staining was determined by using conventional methodology. Examination of cell morphology and cell-wall structure was also made on ultrathin sections by using transmission electron microscopy (Philips 400) after staining with uranyl acetate and lead citrate (Bernalier et al., 1996). The presence of catalase and cytochrome oxidase was examined using standard methods. The optimal growth temperature of strain CRE21T was determined in semi-synthetic BC medium containing glucose (2 g l–1) at pH 6.8 over a temperature range of 25–45 °C (at 1 °C intervals). The influence of pH on growth of CRE21T was evaluated in semi-synthetic BC medium containing glucose (2 g l–1) at 37 °C. The pH range studied was 5.5–7.5 (at 0.1 pH unit intervals, adjusted with varying concentrations of NaHCO3 in the medium).
Cells of strain CRE21T were rods with rounded ends, with a mean length of 1.7 µm and mean width of 0.9 µm. Cells occurred singly or in pairs. Cells stained Gram-negative and thin sections examined via transmission electron microscopy showed a Gram-negative cell-wall structure. Negatively stained cells revealed the absence of flagella. Viable cells could not be recovered from cultures held at 100 °C for 10 min and no spores were observed in cellulose- or in glucose-grown cultures incubated at 37 °C for 30 days or more. Strain CRE21T was strictly anaerobic and required an O2-free medium at a redox potential sufficient to decolorize resazurin (E0=–50 mV). The cells did not possess catalase or cytochrome oxidase activity. With glucose as substrate, the optimal growth temperature was 37 °C with growth occurring from 30 to 39 °C. Strain CRE21T grew from an initial pH of 6.5 up to 7.2, with optimal growth at pH 6.8. These optimal conditions of growth are in line with those found in the human colon. Rumen fluid was not required for growth.
DNA extraction, PCR and sequencing of the 16S rRNA gene of strain CRE21T were performed as reported by Bernalier et al. (1996). Strain CRE21T was grown for 24 h in 50 ml BC medium with glucose (2 g l–1) as carbon source. Cells were harvested by centrifugation for 15 min at 9000 g at 4 °C. The bacterial pellet was then subjected to DNA extraction (Easy DNA kit Genomic DNA Isolation; Invitrogen BV). The 16S rRNA gene was then amplified using the universal primers F8 (5'-AGAGTTTGATCMTGGCTC-3') and 1492R (5'-GNTACCTTGTTACGACTT-3'). Approximately 50 ng of purified PCR product was included in a 20-µl sequencing reaction. Sequencing reactions were performed using a Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer) according to the manufacturer's specifications. All reactions were carried out with an ABI Prism cycle sequencing kit and gels were run on an ABI PRISM 310 automated sequencer. The closest known relatives of the new isolate were determined by performing database searches by using the program FASTA (Pearson & Lipman, 1985). These sequences and those of other related strains were retrieved from GenBank and aligned with the newly determined sequences according to the program SEQtools (http://www.seqtools.dk). The resulting multiple sequence alignment was corrected manually by using the program GeneDoc (Nicholas et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) with the programs SEQtools and TREEVIEW (Page, 1996). The G+C content of the genomic DNA was determined by HPLC by the Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany).
The 16S rRNA gene of strain CRE21T was sequenced (1422 bases) and phylogenetic analysis revealed that this cellulolytic bacterium was most closely related to members of the phylum Bacteroidetes. Strain CRE21T shared 97–98 % 16S rRNA gene sequence similarity with its most closely related species, B. intestinalis, and only 92 % with B. helcogenes. A phylogenetic tree showing the relationships of strain CRE21T to other Bacteroides species is available as Supplementary Fig. S1 in IJSEM Online. The G+C content of the DNA of strain CRE21T was 41.1 mol%; reported values for B. intestinalis and B. helcogenes are 44 and 45 mol%, respectively (Table 1).
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). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) and modified by Huß et al. (1983) by using a model Cary 100 Bio UV/VIS spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in situ temperature probe. Strain CRE21T showed DNA–DNA relatedness of 40 % with B. intestinalis DSM 17393T. This low DNA–DNA relatedness value (<70 %) with its most closely related species indicated that strain CRE21T represented a novel species.
Utilization of different substrates by strain CRE21T was determined with semi-synthetic BC medium containing several carbon sources. Carbon sources were added from sterile stock solutions to a final concentration of 10 mM. Carbon source utilization by strain CRE21T was determined after incubation of the cultures at 37 °C for at least 4 days and by examination of the ability of the strain to maintain growth after three successive transfers on the same substrate. Bacterial growth was monitored by determining the optical density of the culture at 600 nm (OD600). Other biochemical and enzymic activity tests were performed by using the API Rapid ID 32AN and API ZYM test kit systems (bioMérieux) according to the manufacturer's instructions and anaerobic incubation at 37 °C. Furthermore, the cellular fatty acid profile of strain CRE21T was determined using gas chromatography at the Culture Collection, University of Göteborg (Sweden). End products of cellulose and glucose fermentation were determined in 12-day and 24-h cultures, respectively. Gases in the headspace of cultures and short-chain fatty acids in culture supernatants were analysed by gas phase chromatography (Robert et al., 2001). Formate, succinate, ethanol and lactate production were measured using enzymic methods (Roche).
Strain CRE21T was able to utilize a variety of substrates. Sugar utilization and enzymic activity patterns of strain CRE21T were similar to those of B. intestinalis. However, strain CRE21T could be differentiated from B. intestinalis and B. helcogenes based on several characteristics (Table 1
). The major cellular fatty acid of strain CRE21T was anteiso-C15 : 0 (37.8 %) in agreement with data for the genus Bacteroides (Miyagawa et al., 1979) (Table 1). Other cellular fatty acids found in significant amounts in strain CRE21T included iso-C17 : 0 3-OH (12.7 %) and C15 : 0 (15 %). The hydroxy acid iso-C17 : 0 3-OH was also found in B. intestinalis and B. helcogenes but at a higher proportion of the total (19.2 and 17.7 %, respectively). The other cellular fatty acids found in B. intestinalis and B. helcogenes were different from those found in strain CRE21T (Table 1). The end products of cellulose and glucose fermentation by strain CRE21T were mainly acetate, propionate and succinate, with formate and lactate also being produced in smaller quantities (results are given in Supplementary Table S1 in IJSEM Online). Other Bacteroides species isolated from the human gut such as B. ovatus, Bacteroides thetaiotaomicron and B. fragilis similarly produced acetate, propionate and succinate from sugar fermentation (Salyers et al., 1981) but, in contrast to strain CRE21T, they did not produce lactate.
The ability of strain CRE21T and of B. intestinalis DSM 17393T to degrade the main polysaccharides that make up dietary fibres was compared. Strain CRE21T and B. intestinalis DSM 17393T were cultivated in liquid BC medium (10 ml per tube) containing Avicel pH 101 cellulose (100 mg), Sigmacell type 101 cellulose (100 mg), oat spelts xylan (100 mg), pectin from citrus (20 mg), starch from potatoes (20 mg) or CWS (100 mg) as sole energy source. Cultures were incubated at 37 °C for 2–4 days and three culture tubes were inoculated for each substrate. After incubation, substrate utilization was estimated for the two strains by measuring bacterial growth (OD600). In cellulose-, xylan- and CWS-grown cultures, substrate disappearance was measured (disappearance of dry matter) and CMCase and xylanase activities were detected by using an agar plate test (Forano et al., 1994). Cellulase activities of strain CRE21T were further determined by using enzymic assays in which reducing sugars released from carboxymethylcellulose and Avicel pH 101 cellulose were measured colorimetrically with glucose as standard (Robert & Bernalier-Donadille, 2003). Each enzymic assay was performed in triplicate.
Strain CRE21T and B. intestinalis DSM 17393T showed different capacities to utilize dietary polysaccharides (Table 2). Strain CRE21T was able to degrade different types of cellulose (results are shown in Supplementary Fig. S2 in IJSEM Online), including the CWS cellulose, and expressed CMCase [54.0±0.2 µg glucose min–1 (mg protein–1)] and avicellase [24.0±0.1 µg glucose min–1 (mg protein–1)] activities in cellulose-grown cultures. B. intestinalis DSM 17393T was not able to grow on cellulosic substrates. Nevertheless, B. intestinalis showed the ability to degrade xylan whereas poor growth and weak xylanase activity were detected for strain CRE21T. The two bacterial taxa were able to use pectin and starch but B. intestinalis showed poor ability to degrade starch compared with strain CRE21T. Polysaccharide utilization profiles thus differentiated between strain CRE21T and B. intestinalis DSM 17393T. Strain CRE21T remains the sole Bacteroides representative isolated from the human gut and described to date that is able to degrade cellulose.
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), saccharolytic, producing acetate and succinate as the major metabolic end products (Holdeman & Moore, 1974) and anteiso-C15 : 0 as the major cellular fatty acid (Miyagawa et al., 1979). Strain CRE21T was most closely related to B. intestinalis, which was isolated from the human colon, but these two taxa showed a low level of DNA–DNA relatedness (<70 %). In addition, phenotypic differences could be shown between strain CRE21T and B. intestinalis, in particular in their abilities to degrade polysaccharides. Strain CRE21T is characterized by its ability to degrade cellulose compared with B. intestinalis as well as other Bacteroides species described from the human gut. Given this physiological characteristic, we propose the name Bacteroides cellulosilyticus sp. nov. to accommodate strain CRE21T.
Description of Bacteroides cellulosilyticus sp. nov.
Bacteroides cellulosilyticus (cell.u'lo.si.ly'ti.cus. N.L. n. cellulosum cellulose; Gr. adj. lutikos loosening, dissolving; N.L. adj. lyticus -a -um dissolving; N.L. masc. adj. cellulosilyticus cellulose-dissolving).
Cells are non-motile rods, 1.7 µm in lengh and 0.9 µm in width. Gram-negative by staining and cell-wall ultrastructure. No heat-resistant endospores are formed. Strictly anaerobic. Colonies developed on glucose-BC agar medium are white to slightly brown, translucent, circular with entire margins and about 2–5 mm in diameter. Cytochrome oxidase- and catalase-negative. Metabolizes cellulose to acetate, propionate and succinate. Also ferments glucose, sucrose, fructose, maltose, xylose, galactose, ribose, melibiose, mannose, lactulose, galacturonic acids, pectin, starch and cellulose. Poor growth is exhibited with lactose, raffinose, arabinose, cellobiose, aesculin, xylan and salicin. No growth is observed on trehalose, mannitol, inositol, sorbitol or fucose. Positive reactions are obtained with the API Rapid ID 32AN and API ZYM systems for N-acetyl-
-glucosaminidase, alkaline phosphatase, alanine arylamidase, 
-arabinosidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, leucyl glycine arylamidase, mannose, raffinose, indole -fucosidase (weak), phenylalanine arylamidase (weak), tyrosine arylamidase (weak), acid phosphatase, naphthol-AS-BI-phosphohydrolase and esterase lipase C8 (weak). Growth occurs between 30 and 39 °C, and pH 6.5 and 7.2. Optimal growth conditions are 37 °C and pH 6.8. Rumen fluid is not required for growth. Major fatty acids are anteiso-C15 : 0 (37.8 %), iso-C17 : 0 3-OH (12.7 %) and C15 : 0 (15 %). The G+C content of the DNA of the type strain is 41.1 mol%.
The type strain, CRE21T (=DSM 14838T=CCUG 44979T), was isolated from human faeces of a non-methane-excreting individual.
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ACKNOWLEDGEMENTS |
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