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1 Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury SP4 0JG, UK
2 Rumen Biotechnology, AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand
3 Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand
Correspondence
Paul W. Riley
paul.riley{at}hpa.org.uk
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Present address: Infection Control Team, Health Protection Scotland, 1 Cadogan Square, Cadogan Street, Glasgow G2 7HF, UK. 
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GPC 589T is DQ925472.
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), it is an important source of ammonia especially in the recycling of nitrogen in the rumen. While urea is physiologically important to nitrogen metabolism in ruminants, it is also economically important to ruminant feeding. However, the microbial aspects of urea metabolism are not well understood, either regarding numbers relative to total rumen bacteria or whether the ureolytic bacteria are widespread in rumen contents, or even whether bacteria are the major source of rumen urease (Farlin et al., 1968).
Strain GPC 589T was isolated from the rumen fluid of a penned cannulated sheep that had been fed on a diet of hay and grass with water ad libitum. The anaerobic culture technique of Hungate (1950) was used with oxygen-free CO2 as the gas phase and incubation at 37 °C. Initially, a fresh rumen fluid sample (0.5 %, v/v) was enriched for 48 h (Cook, 1976) in medium GM comprising (w/v): Bacto casitone (0.5 %), Bacto yeast extract (0.5 %), KH2PO4 (0.25 %), Na2HPO4 (0.25 %), CH3 . COONa (0.1 %), mannitol (0.2 %), MgCl2 . 6H2O (0.05 %), sodium thioglycollate (0.075 %) and resazurin (0.0001 %), pH 7.0; autoclaved at 103.5 kPa for 15 min. Filter-sterilized urea (0.2 % w/v, final concentration) was added to cooled medium. The enrichment was maintained by overnight subculturing (0.1 ml) in 8 ml medium G (mannitol-free GM) in Hungate tubes. An ureolytic Gram-positive coccus was isolated from a Hungate roll tube of medium GM (4.5 ml containing 2 % agar) that had been inoculated (0.5 ml) from the 10–7 dilution of a decimal dilution series (medium G) of an overnight subculture; this was identified presumptively as an enterococcus. After overnight incubation (about 18 h) in medium G, a decimal dilution series showed a hazy growth in the 10–9 dilution in contrast to the heavy growth present at lower dilutions: there was no visible growth in the higher dilutions. Over a 3-day incubation period during which time the ureolytic activity of this 10–9 dilution culture increased, there was no discernible increase in turbidity. The Gram-positive isolate from the 10–9 dilution tube was designated strain GPC 589T. Urease activity was determined by measuring ammonia production in the presence and absence of urea. A 1 ml centrifuged sample of a broth culture was resuspended in an equal volume of 20 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA. A 25 µl sample was incubated with 0.17 mmol urea in a final volume of 1 ml for 5 min at 37 °C. The reaction was terminated by the addition of phenate-hypochlorite reagents (Weatherburn, 1967), modified such that 1 ml of each reagent was used in an assay. The colour was developed for 25 min at 37 °C and read at 570 nm. Culture growth was measured by determining the viable count as turbidities were too low. Viable counts of cultures were carried out within the oxygen-free atmosphere of a flexible-film anaerobic chamber, using procedures described by Hartley et al. (1992). BHF plates of supplemented brain heart infusion blood agar (Barr et al., 1987), stored anaerobically (10 % CO2, 10 % H2 in N2, >90 % RH) at 4 °C, were lightly air-dried at 37 °C before being transferred to the anaerobic chamber for overnight storage. The source of brain heart infusion used in this medium (Oxoid) was found to be important: other sources were inferior (data not shown). The agar medium was inoculated with triplicate 25 µl samples of a decimal dilution series of a culture prepared in brain heart infusion broth using a modification of the technique of Miles et al. (1938), as described previously by Hudson et al. (1984). The plates were removed from the chamber and incubated anaerobically at 37 °C for 72 h (Hartley et al., 1992). Gram staining was based on the method of Preston & Morrell (1962) but with modifications to the iodine mordant (Magee et al., 1975), the safranin counterstain (Holdeman et al., 1977) and the use of acetone to fix the air-dried slides (Mangels et al., 1984). Following publication of the procedure of Johnson et al. (1995), staining was done anaerobically to ensure uniformity of stain. Cell morphology was observed using a Leitz Dialux light microscope at a magnification of x1000 with cells grown in medium G overnight at 37 °C.
Strain GPC 589T was characterized biochemically by using a combination of conventional tests as described in the VPI Anaerobe Laboratory Manual (Holdeman et al., 1977). All biochemical tests were performed in triplicate. The following compounds did not augment the growth of strain GPC 589T when added to medium G and grown anaerobically at 37 °C: carbohydrates: glucose, sucrose, maltose, starch, mannitol and cellobiose; carboxylic compounds: pyruvate, lactate, malate, formate, fumarate, succinate and citrate; Tween 80; mixture of volatile fatty acids (propionic, n-butyric, n-valeric, iso-butyric, 3-methylbutyric and 2-methylbutyric; Holdeman et al., 1977); nitrogen compounds (urea omitted): ammonia and nitrate; haemin; serum; vitamin K and bicarbonate. The following were required for optimum growth: casitone, yeast extract, acetate, magnesium, sodium/potassium phosphate, thioglycollate and urea. Thioglycollate was required for a reduced environment; cysteine was inhibitory. Acetate stimulated growth. Urea was demonstrated to be essential for growth of strain GPC 589T; the minimal growth in medium without urea (Bacto casitone is a milk product and contains trace amounts of urea) contrasts with the 140-fold increase in the presence of urea at 0.2 % (w/v). Whereas urea was essential for growth, ammonia, a product of the strong urease activity, did not support growth. The role for the hydrolysis of urea was to generate ATP. Thus, preincubation of strain GPC 589T with the urease inhibitor fluorofamide (Millner et al., 1982; Kenny, 1983) resulted in an 87 % inhibition of ammonia production and a concomitant 92 % reduction in ATP synthesis. Strain GPC 589T did not grow at 20 or 50 °C. The G+C content of the DNA was determined by HPLC as described by Ezaki et al. (1990). DNA from peptostreptococci (Peptostreptococcus asaccharolyticus, Peptostreptococcus prevotii and Peptostreptococcus magnus), ruminococci (Ruminococcus torques and Ruminococcus obeum) and Peptococcus niger was used for reference. The G+C content of the DNA of strain GPC 589T was found to be 34 mol%, which was consistent with the assignment of this isolate to the low G+C branch of the Gram-positive bacteria.
Fatty acid methyl esters were obtained from 40 mg cells scraped from Petri dishes by saponification, methylation and extraction, using minor modifications of the methods of Miller (1982) and Kuykendall et al. (1988). The fatty acid methyl ester mixtures were separated using the Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID), which consisted of an Agilent model 6890N gas chromatograph fitted with a 5 % phenyl-methyl-silicone capillary column (0.2 mmx25 m), a flame-ionization detector, Agilent model 7683A automatic sampler and an HP-computer with MIDI database (Hewlett Packard Co.). Peaks were integrated automatically and fatty acid names and percentages were calculated using the MIS standard software (Microbial ID). The gas chromatographic parameters were as follows: carrier gas, ultra-high-purity hydrogen; column head pressure, 60kPa; injection volume, 2 µl; column split ratio, 100 : 1; septum purge, 5 ml min–1; column temperature, 170–270 °C at 5 °C min–1; injection port temperature, 240 °C; and detector temperature, 300 °C (Kämpfer & Kroppenstedt, 1996). The cellular fatty acid composition of strain GPC 589T consisted of straight-chain saturated and unsaturated fatty acids. The principal fatty acids were the saturated acids C16 : 0 (29.2 %) and C18 : 0 (40.7 %). There were also smaller amounts of the saturated fatty acids C14 : 0 (0.7 %) and C17 : 0 (1.7 %). The unsaturated acids C16 : 1
9c (2.4 %), C18 : 19c (4.5 %) and C18 : 111c (1.1 %) were also present. An unidentified compound with an equivalent chain-length (ECL) of 13.523 was detected (19.7 %), but could not be assigned to a known fatty acid or a dimethylacetal.
The 16S rRNA gene sequence was determined from the isolated DNA (Luton et al., 2002) using the primers of Hutson et al. (1993) and PCR was performed using the protocol of Innis & Gelfand (1990). The 16S rRNA gene was amplified by PCR using the universal eubacterial primers (Lane, 1991). Sequenced 16S rRNA gene fragments were assembled into one contiguous sequence. Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and TreeView (Page, 1996), after alignment of data using ARB (Ludwig et al., 2004). Distances (Kimura two-parameter model) and clustering by neighbour-joining methods were determined by using bootstrap values based on 1000 replicates (Fig. 1). The 16S rRNA gene sequence of strain GPC 589T was a continuous stretch of 1531 bp. Sequence database searches showed that strain GPC 589T displayed the highest sequence relatedness to members of the Clostridium subphylum (data not shown), consistent with the low G+C content. On the basis of sequence similarities, the closest named relatives of strain GPC 589T corresponded to species within Clostridium rRNA cluster XIVa (see Collins et al., 1994, for rRNA group designations). Sequence similarity calculations indicated that the closest relatives of strain GPC 589T were Catonella morbi ATCC 51271T (86.5 %), Johnsonella ignava ATCC 51276T (85.3 %) (these were placed in cluster XIVa by Willems & Collins, 1995), Eubacterium saburreum C27KA (87.4 %), Clostridium polysaccharolyticum DSM 1801T (88.3 %), Acetitomaculum ruminis 139BT (87.2 %) and Eubacterium hallii ATCC 27751T (87.5 %). These bacteria were isolated from such diverse habitats as the human buccal cavity, the rumen and human faeces. The monospecific genera Catonella and Johnsonella, described by Moore & Moore (1994), were isolated from the human gingival crevice and periodontal pockets (Moore et al., 1985) and were originally referred to as ‘Bacteroides D42’ and ‘Bacteroides D19’, respectively. Holdeman & Moore (1974) described E. hallii, which was isolated from human faecal flora. A. ruminis was isolated from the bovine rumen and described by Greening & Leedle (1989). In 1980, van Glyswyk isolated Fusarium polysaccharolyticum from the sheep rumen, which was later renamed Clostridium polysaccharolyticum (van Gylswyk et al., 1980). The description of the anaerobic filamentous micro-organisms isolated from human dental plaque and gingival crevices but unnamed by Theilade & Gilmour (1961) was used by Holdeman et al. (1977) in their description of E. saburreum (Skerman et al., 1980). A BLAST search using the 16S rRNA gene of strain GPC 589T revealed that uncultured bacterial clones have been identified from humans, which are closer to strain GPC 589T than the species in Clostridium rRNA cluster XIVa. Ley et al. (2006) identified two clones, RL249 (GenBank accession no. DQ805137) and RL181 (DQ798309) from human faeces and Eckburg et al. (2005) identified clone MH95 (AY982262) from a biopsy of the human colon mucosa and a further three clones from biopsies of the human colon, MG76 (AY982192), LY31 (AY976352) and MG71 (AY916289).
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). Phylogenetically, strain GPC 589T has an affinity with rRNA cluster XIVa, but not with the Gram-positive anaerobic cocci that are interspersed throughout a variety of phylogenetic groups. The association of strain GPC 589T with members of Clostridium rRNA group XIVa was only loose, with strain GPC 589T branching deeply at the periphery of the cluster. Treeing analysis showed that strain GPC 589T did not have a close relationship with any individual member of Clostridium rRNA group XIVa, although the branching at the base of the grouping was supported by a high bootstrap value. The distinctiveness of strain GPC 589T was also evident from divergence values of greater than 12 % with members of rRNA cluster XIVa and of greater than 20 % with other recognized Gram-positive anaerobic cocci.
Clostridium rRNA cluster XIVa embraces a diverse range of organisms including Catonella morbi, J. ignava, E. hallii, A. ruminis, E. saburreum and a variety of clostridial species, Clostidium aerotolerans, Clostridium oroticum, Clostridium polysaccharolyticum, Clostridium proteolyticum, Clostridium ramosum and Clostridium formicaceticum. In addition to high sequence divergence values and tree topology considerations, support for the separateness of strain GPC 589T from other organisms within Clostridium rRNA cluster XIVa also comes from phenotypic considerations (Table 1). In particular, strain GPC 589T can be readily distinguished from all other taxa within this cluster by its small, coccoid cells and the synthesis of ATP from the hydrolysis of urea. Furthermore, strain GPC 589T can be distinguished from Clostridium polysaccharolyticum by not producing endospores. The non-fermentative characteristic distinguishes strain GPC 589T from Catonella morbi, E. hallii, E. saburreum and A. ruminis. The phenotypic distinctiveness of strain GPC 589T, together with its long rRNA line branching at the base of Clostridium rRNA cluster XIVa, merits its inclusion in a new genus and species, Howardella ureilytica gen. nov., sp. nov.
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Gram-positive-staining, non-spore-forming, non-motile, coccoid cells. Growth occurs under strictly anaerobic conditions. Carbohydrates are not fermented. Nitrate is not reduced. Acetate stimulates growth. Member of the Clostridium subphylum of the Gram-positive bacteria and is phylogenetically loosely associated with Clostridium rRNA group XIVa. The type species is Howardella ureilytica.
Description of Howardella ureilytica sp. nov.
Howardella ureilytica (u.re.i.ly'ti.ca. N.L. n. urea urea; Gr. adj. lutikos able to dissolve; N.L. adj. lyticus -a -um dissolving; N.L. fem. adj. ureilytica urea dissolving).
Exhibits the following properties in addition to those given in the genus description. Cells are 0.77–0.83 µm longx0.61–0.67 µm wide and occur singly, in pairs, although occasionally in short chains of 6–8 cells. After 72 h anaerobic incubation at 37 °C under a gas phase of N2, CO2 and H2 (80 : 10 : 10, by vol.) on BHF/blood agar, colonies are minute to 1 mm in diameter, circular, entire, domed and white. Urea is obligatory. Carbohydrates are not fermented. Hydrolysis of urea by cytostolic urease generates ATP. Long-chain cellular fatty acids consist of a complex mixture including an unknown with an ECL of 13.523, C16 : 0 and C18 : 0. The G+C content of the DNA of the type strain is 34 mol%. Habitat, gastrointestinal tract.
The type strain, GPC 589T(=DSM 15118T=JCM 13267T), was isolated from a sheep rumen.
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ACKNOWLEDGEMENTS |
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