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Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Correspondence
R. J. Wallace
rjw{at}rri.sari.ac.uk
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The GenBank accession number for the 16S rDNA sequence of strain I-6T is AJ310135.
Present address: Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido, Japan. 
Present address: Microbiology Section, Animal Nutrition Division, IVRI, Izatnagar-243 122, India.
Present address: Danisco Cultor Innovation, FIN-02460, Kantvik, Finland.
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). So-called ‘ammonia-hyperproducing’ (also referred to as ‘hyper-ammonia-producing’ or HAP) bacteria, non-saccharolytic amino acid fermenters and rapid producers of ammonia from amino acids, are partly responsible for this inefficiency (Chen & Russell, 1988, 1989, 1990; Paster et al., 1993; Russell et al., 1988, 1991). The first HAP species isolated were Clostridium aminophilum, Clostridium sticklandii and Peptostreptococcus anaerobius (Chen & Russell, 1988, 1989; Paster et al., 1993; Russell et al., 1988, 1991). Subsequently, similar, though not the same, HAP bacteria were isolated from cattle in New Zealand (Attwood et al., 1998) and Australia (McSweeney et al., 1999). This paper describes the isolation and properties of a HAP bacterial species isolated from sheep in the UK. It differs significantly from the other HAP species, not only phylogenetically but in its formation of higher levels of volatile fatty acids (VFAs) from acetate and propionate.
Isolation of bacteria and growth conditions
Strains I-6T and I-8 were isolated from a 10-6 dilution of ruminal fluid from a sheep by virtue of their ability to grow anaerobically on the defined medium of Chen & Russell (1988) containing trypticase peptone (15 g l-1; Becton Dickinson Microbiology Systems) but no other energy source (CRT medium). The sheep received a maintenance diet of hay, barley, molasses, fish-meal and vitamins/minerals [500, 299·5, 100, 91 and 9·5 g (kg dry matter)-1, respectively] twice daily. A sample of ruminal fluid was removed 3 h after feeding, kept warm and under CO2, and strained through four layers of muslin. Strained rumen fluid was diluted serially under CO2 in basal CRT medium without added trypticase. The medium was adjusted to pH 7·0 before autoclaving. These dilutions were used to inoculate (1 %, v/v) Hungate tubes containing CRT medium. The cultures were incubated at 39 °C for 7 days, and 20 µl of the 10-6 dilution was then streaked cross-wise onto plates of CRT medium and incubated again for 7 days. Two colonies were picked and grown in the liquid form of ruminal fluid-containing medium M2 (Hobson, 1969); bacteria were then reisolated by repeating the streaking and colony selection on CRT plates. Isolates were stored in medium M2.
Bacterial identification
The bacteria were characterized using conventional methods based on those of Holdeman et al. (1977). The presence of spores was investigated by incubating overnight M2 cultures at 80 °C for 10 min and then reinoculating fresh tubes of M2 liquid medium. Fermentation products were determined by derivatization and capillary GLC of supernatants from cultures grown in liquid M2 (Richardson et al., 1989). Some fermentation tests were carried out using API 20 A strips (bioMérieux) under anaerobic conditions to determine sugar utilization and indole production. Gelatin liquefaction was assessed as described by Holdeman et al. (1977) except that the liquid form of medium M2 (Hobson, 1969) was the basal medium. Sugar utilization was also tested by adding the substrate to CRT medium to a final concentration of 5 g l-1 and determining the growth response turbidimetrically at 650 nm using a Novaspec II spectrophotometer (Amersham Pharmacia Biotech). Staining for cytoplasmic inclusions and chemical analysis for storage polysaccharide and poly-
-hydroxybutyrate were carried out as described by Hanson & Phillips (1981). The G+C content was determined by means of the differential dye-binding method of Apajalahti et al. (2001), using regression analysis (r2>0·99) of data obtained from gradients containing standard DNA samples of known G+C content (Clostridium perfringens, Escherichia coli and ‘Micrococcus lysodeikticus’). Sample DNA was analysed in triplicate. SDS-PAGE was carried out using pre-cast acrylamide gradient gels (ExcelGel SDS, gradient 8–18 %T; Amersham Pharmacia Biotech). Whole cells were sedimented from liquid M2 cultures by centrifugation (at 15 000 g for 15 min) and the pellet was resuspended in SDS sample buffer, boiled and then centrifuged once more (at 12 000 g for 10 min) before being applied to the gel.
Ammonia production rate
The method used here was based on that described by Russell et al. (1988). Bacteria were grown overnight in 10 ml CRT medium; 1 ml was removed anaerobically into a microcentrifuge tube on ice and centrifuged (12 000 g for 10 min), 1 ml 150 g trypticase l-1 was added to the remaining 9 ml in the culture tubes and the tubes were then incubated at 39 °C for 6 h. A further 1 ml sample was taken and centrifuged. Ammonia concentrations were determined for the supernatant fluids by using an automated phenol/hypochlorite method (Whitehead et al., 1967) and protein in the pellet was determined using the Folin reagent following alkaline digestion (Herbert et al., 1971). Ammonia production rates were calculated as the ammonia produced divided by the mean protein concentration in the 0 h and 6 h samples. Results are means obtained from three separate cultures.
Electron microscopy
Bacteria were fixed for 2 h in 2·5 % glutaraldehyde in 0·1 M sodium cacodylate buffer, pH 7·3, then post-fixed for 1 h in 1 % osmium tetroxide in the same buffer. The fixed bacteria were washed in the cacodylate buffer and embedded in 1·5 % agarose. Small blocks of the agarose-embedded sample (1 mm2) were dehydrated in a graded ethanol series, cleared in propylene oxide and infiltrated overnight in a 50 : 50 mixture of propylene oxide and Araldite resin (Agar Scientific). The infiltrated specimens were embedded in fresh Araldite resin and the blocks were polymerized for 3 days at 70 °C. Ultrathin sections of embedded samples were contrasted with uranyl acetate and lead citrate and examined in a JEOL 1200 EXB transmission electron microscope operating at an accelerating voltage of 80 or 100 kV. Whole bacteria were also examined in the electron microscope after negative staining in 2·5 % ammonium molybdate, pH 6·5.
rDNA analysis
DNA from strain I-6T was extracted using methods described by McEwan et al. (1994). A partial sequence of 16S rDNA was amplified using the universal primers 5'-AGAGTTTGATCATGGCTCAG-3' and 5'-ACGGCTACCTTGTTACGACTT-3' (Weisburg et al., 1991) in a mixture that contained 1 µM each primer, 200 µM each dNTP, 50 mM KCl and 1·5 M MgCl2 in 10 mM Tris/HCl (pH 8·3) buffer. Next, 0·1 µg DNA and 2·5 U Taq DNA polymerase were added to 100 µl of this mixture and amplification was carried out using 45 cycles of 94 °C for 1 min followed by 2 min at 55 °C and 3 min at 72 °C. Amplification was confirmed on an agarose gel.
Amplified DNA was cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions. Plasmids were isolated from recombinant colonies (Maniatis et al., 1982) and the nucleotide sequence of the insert was determined using an ABI Prism 377XL DNA sequencer (Perkin Elmer). A DNA similarity search was performed using the DDBJ BLAST server (http://www.ddbj.nig.ac.jp/E-mail/homology.html). The phylogenetic tree was constructed using CLUSTAL W analysis (http://www2.ebi.ac.uk/clustalw/) followed by programs within PHYLIP version 3.57 (Felsenstein, 1989). Analysis within PHYLIP was performed by first using the DNADIST and NEIGHBOR programs. The validity of the resulting tree was determined using 1000 bootstrap iterations (SEQBOOT, DNADIST, NEIGHBOR and CONSENSE programs). The initial tree was viewed using TreeView (Page, 1996) and bootstrap values inserted using FREEHAND (version 9).
Identification and phylogenetic analysis
Whole-cell extracts from isolates I-6T and I-8 produced identical banding patterns in SDS-PAGE. The patterns were distinct from banding patterns of other ruminal bacteria, including other non-saccharolytic Clostridium/Eubacterium species and the HAP species identified previously, P. anaerobius, C. sticklandii and C. aminophilum (results not shown). The morphology of isolate I-6T was typical of a Gram-positive organism, having dimensions of approx. 1·2x0·4 µm and a thin, layered cell wall (Fig. 1). More distinctive were inclusions of low electron density, which appeared under transmission electron microscopy (Fig. 1a, b), and membrane-like structures associated with the septum of dividing cells and in a polar location in non-dividing cells (Fig. 1c, d). The former gave the appearance of storage materials, but staining and chemical analysis for glycogen and poly--hydroxybutyrate did not give positive results.
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) and similar to the G+C values found for C. aminophilum, another ruminal HAP species (Paster et al., 1993).
Isolates I-6T and I-8 were unusual in their VFA metabolism. During growth on M2 medium, which contains clarified ruminal fluid and therefore VFAs, there was a consistent decrease in the concentrations of acetate and propionate during growth (Table 1). Caproate was the major product and, to a lesser extent, valerate was also formed. The concentration of caproate formed was higher than any we have encountered previously with any ruminal species and the utilization, rather than production, of propionate is, as far as we are aware, unique among ruminal species of bacteria.
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; Fig. 2). It represents a novel species, located on a different branch with respect to the original HAP bacteria isolated by Russell and his colleagues (Fig. 2). Isolate I-6T is most closely related, among published isolates, to HAP isolate C2 of Attwood et al. (1998) (89 % identity), two asaccharolytic species, Eubacterium minutum and Eubacterium infirmum, from the mouth (Cheeseman et al., 1996; Wade et al., 1999a, b) (also 89 % identity), and another oral species, Eubacterium brachy (Hamid et al., 1994) (88 % identity). Its 16S rDNA sequence is very similar to three sequences obtained from the rumen by Tajima et al. (2000) (accession nos AB034093, AB034092 and AB034148). These bacteria do not fall into any of the clusters proposed by Collins et al. (1994), being taxonomically quite distant from the closest group, cluster XI (Attwood et al., 1998; Cheeseman et al., 1996). The bacteria share an asaccharolytic physiology as well as a common location in the digestive tract, suggesting a common progenitor.
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), though not as high as some of the rates, up to 946 nmol (mg protein)-1 min-1, observed with isolates from grazing ruminants in New Zealand (Attwood et al., 1998).
Ecological niche
Strain I-6T thus appeared to be typical of the non-saccharolytic HAP bacteria isolated from the rumen first by Russell and his colleagues (Chen & Russell, 1988, 1989; Paster et al., 1993; Russell et al., 1988) and subsequently by Attwood et al. (1998) and McSweeney et al. (1999). It was similar to these other isolates in its ability to grow on trypticase in the absence of sugars, its non-saccharolytic metabolic properties and its sensitivity to monensin. Like most of the isolates obtained in this way, with the exception of the Clostridium botulinum-like isolate Lp1284 (McSweeney et al., 1999), it was not proteolytic. Most importantly from the point of view of ruminal ecology, isolate I-6T produced NH3 rapidly from amino acids. Thus, despite the relatively small numbers of these bacteria, representing around 1 % of the population, they may still play a significant role in ruminal ammonia formation (Russell et al., 1991).
Phylogenetic analysis based on rDNA similarity indicated that strain I-6T was a novel species. Strain I-6T was metabolically and morphologically similar to two other groups of Clostridium/Eubacterium species isolated from sheep on the same diet, but phylogenetically separate and having different whole-cell SDS-PAGE banding patterns (S. C. P. Eschenlauer, N. McKain, N. D. Walker, N. R. McEwan, C. J. Newbold and R. J. Wallace, unpublished data). Isolate I-6T grew rapidly on pyruvate, in contrast to related species and other HAP bacteria, which were reported to ferment pyruvate only weakly (Attwood et al., 1998) or not at all (Chen & Russell, 1989; Russell et al., 1991). Pyruvate is not present at high concentrations in ruminal fluid (Wallace, 1978). The ability of strain I-6T to grow rapidly on pyruvate presumably reflects only the central role of pyruvate in metabolism. The niche that strain I-6T occupies may be one of a scavenger, converting amino acids and other compounds formed as the result of fermentation of feedstuffs and autolysis of other rumen microbial species to pyruvate to generate energy via the oxidation of pyruvate to acetate. The interconversion of VFAs to form caproate may be a mechanism for regenerating oxidized cofactors to enable the fermentation to proceed, a scenario similar to that observed in Clostridium kluyveri (Smith et al., 1985).
Description of Eubacterium pyruvativorans sp. nov.
Eubacterium pyruvativorans (pyr.uv.at'i.vor.ans. N.L. n. pyruvatum pyruvate; L. v. vorans devouring, eating greedily; N.L. neut. adj. pyruvativorans devouring pyruvate).
Cells are Gram-positive, straight to slightly curved rods, 0·3–0·5 µm wide and 1·0–1·5 µm long. Cells occur in short chains. No flagella are present and spores are not evident. The G+C content of the DNA is 56·8 mol%. Heating in the spores test eliminates viable cells. Overnight growth in M2 broth produces a uniformly opalescent suspension. Colonies on M2 solid medium are, after 72 h, light tan in colour and 2 mm in diameter and have slightly irregular edges. Growth is supported by pyruvate and, to a lesser extent, lactate. Amino acids can be used as the sole source of carbon and energy, with yields much lower than those obtained with pyruvate. Sugars are not fermented. No growth occurs aerobically. The main fermentation product is caproate in rich, ruminal fluid-containing medium.
The type strain, I-6T (=ATCC BAA-574T=NCIMB 13911T), was one of two isolates obtained from the sheep rumen using a medium selective for bacteria that can grow on peptides as the sole source of carbon and energy. The other strain, I-8, had an identical whole-cell protein SDS-PAGE pattern, similar fermentation activities and products and similar NH3-producing activity with respect to strain I-6T.
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ACKNOWLEDGEMENTS |
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