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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences. Beijing 100080, PR China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) is a group of Gram-positive, asporogenous, non-acid-fast bacteria and cells are irregular rods, filamentous, diphtheroidal or branching. Species of the genus are either facultative or obligate anaerobes. Members of the genus Actinomyces primarily inhabit the mucosal surfaces of human and animals, such as the oral cavity (Schaal, 1986, 1992). In recent years, several novel species of Actinomyces have been isolated from human and animal sources (Hall et al., 2003a, b, c, 2005; Hoyles et al., 2001a, b, c, 2002; Lawson et al., 2001; Nikolaitchouk et al., 2000; Pascual et al., 1999). While most of these species have been associated with clinical materials, their role in pathogenesis is not known and, at best, these organisms can be described as opportunistic pathogens.
Ruminants harbour numerous micro-organisms in their rumen that endow them with the ability to digest fibrous materials (Chesson & Forsberg, 1997; Koike et al., 2003; Nelson et al., 2003). During a study of rumen micro-organism composition, 10 bacterial strains that degraded xylan efficiently were obtained from a 4-year-old animal of the Jinnan cattle breed (Bos taurus), a local species from northeast China. Two of the novel strains were found to be closely related, phylogenetically and phenotypically, to members of the genus Actinomyces. However, both novel strains differed from recognized species. In this paper, a novel species of the genus Actinomyces is described.
Rumen content, sampled from a 4-year-old castrated bullock within 20 min after slaughter, was inoculated in xylan medium as described by Krause et al. (2001) and incubated under 100 % N2 in anaerobic tubes (18x180 mm) sealed with butyl rubber stoppers at 39 °C for 3 days. After subculturing on xylan medium five times, strains B71T and D471 were isolated from the xylan-degrading mixtures by the Hungate roll tube technique (Hungate, 1969) using the same medium containing 15 % agar. Single colonies were picked and transferred to PYRG broth [peptone-yeast extract-glucose (PYG) + 15 % rumen fluid]. Strains were routinely cultured in PYRG medium unless otherwise indicated.
Cell morphology was examined with a light microscope (BH-2; Olympus) and an electron microscope (H-600A; Hitachi). For electron microscopy, bacterial cells grown at 39 °C for 2 days were coated with a palladium/iridium alloy using a high vacuum evaporator (HUS-5GB; Hitachi). For determination of the end products of glucose fermentation, the novel strains were grown in PYRG at 39 °C for 7 days. Short-chain fatty acids were measured by GC (GC-14B; Shimadzu) using stainless steel columns packed with Porapak GDX-401 (60–80 mesh) and detected with a flame-ionization detector. For measurement of non-volatile fatty acids, methyl esters were derived from samples according to Holdeman et al. (1977) before analyses. Column temperatures were 220 and 150 °C for the measurement of volatile and non-volatile fatty acids, respectively. Nitrogen was used as the carrier gas in all analyses. The presence of formic acid was determined using HPLC (1525; Waters) installed with a Hewlett Packard C18 HPLC column (3.9x150 mm) according to Akalin et al. (2002). Acetonitrile (60 %, v/v) in ultra-purified water was used as the mobile phase and the flow speed was 0.3 ml min–1.
The diagnostic isomers of diaminopimelic acid in the cell-wall peptidoglycan were determined using established TLC procedures (Lechevalier & Lechevalier, 1980). Cellular fatty acids were extracted, methylated and analysed using the standard MIDI system (Miller, 1982; Sasser, 1990). Menaquinones were analysed by using HPLC (Kroppenstedt, 1985) with Micromonospora halophytica DSM 43171T as a reference.
Temperature profiles were determined in PYRG using a water bath at temperatures of 15–55 °C at 1 °C intervals. The pH range for growth was determined in PYRG broth at various pH values adjusted with HCl or NaOH (1 M l–1) at 39 °C. Growth was monitored by measuring the OD600 of cultures at 1, 3 and 7 days. The generation time of the strains was determined by monitoring the OD600 of the PYRG culture at 46 °C and pH 8.5 at 1 h intervals up to 48 h. Biochemical traits were assessed using conventional methods (Holdeman et al., 1977). For determination of xylan degradation, 100 µl xylan culture was dried at 100 °C in a tube with a lid and then hydrolysed into monosaccharide by the addition of 500 µl 0.25 M H2SO4 at 120 °C for 90 min. The hydrolysed mixture was adjusted to neutral pH with 100 µl 10 M NaOH and the reductive carbohydrate was estimated as described by Miller (1959). Starch was determined by using the Congo red overlay method (Ruijssenaars & Hartmans, 2001). All tests were performed in duplicate.
Genomic DNA was extracted and purified using the method of Marmur (1961). The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962) using a spectrophotometer (DU800; Beckman) with Escherichia coli K-12 as the reference. The 16S rRNA gene was amplified by PCR using a pair of universal primers, 27F (5'-AGAGTTTGATCC/ATGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCC-3'), corresponding to base positions 8–27 and 1541–1525 of the 16S rRNA gene of E. coli (Winker & Woese, 1991), respectively. Extracted genomic DNA was used as a template and PCR amplification was performed with Thermolyne Amplitron I (Barnstead Thermolyne). PCR products were purified using a UNIQ-10 PCR product purification kit (Sangon) and ligated into vector pUCm-T (Sangon), as recommended by the manufacturer. Primers T7 (5'-GTAATACGACTCACTATAGG-3') and M13R (5'-CAGGAAACAGCTATGACCAT-3') were used to sequence the 16S rRNA gene fragment. Sequencing was performed by the Sangon Biological Engineering Technology Service, Shanghai, China, using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kits (Perkin Elmer) and an ABI PRISM 377XL DNA sequencer. The 16S rRNA gene sequences of strains B71T and D471 were submitted to GenBank to search for similar sequences using the BLAST algorithm. The best matching sequences were retrieved from the database and aligned. Similarity analysis was performed using CLUSTAL_X (Thompson et al., 1997). Phylogenetic trees were constructed using neighbour-joining and maximum-parsimony methods implemented in the MEGA2 (Kumar et al., 2001) and PHYLIP software packages (Felsenstein, 1993). The resulting tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings.
Strains B71T and D471 were Gram-positive, non-motile, non-spore-forming, straight rods (0.5–1.0x2.5–4.0 µm) and were non-acid-fast. They did not produce catalase and showed strictly anaerobic growth. The novel strains hydrolysed xylan and starch and fermented several kinds of mono-, di- and oligosaccharides. The major end products from glucose fermentation were formic, acetic and lactic acids. The novel strains did not hydrolyse hippurate or urea. Hydrolysis of aesculin and glycogen and fermentation of inositol, mannitol, mannose and salicin were variable between the two isolates. Both novel strains grew at 20–55 °C and at pH 6.5–9.0, with optimum growth at 46 °C and pH 8.0–8.5. The mean generation time for the novel strains was 2.21 h when grown in PYRG at 46 °C and pH 8.5.
To determine the phylogenetic position of the novel strains, their complete 16S rRNA gene sequences were analysed. A phylogenetic tree was constructed based on the similarity of a consensus length of 1367 bp of 16S rRNA gene sequence (Fig. 1). Strains B71T and D471 were found to cluster with other species of the genus Actinomyces. However, the novel strains formed a distinct branch with relatively low sequence similarities (88.3–93.6 %) to other Actinomyces strains. The greatest gene sequence similarity (93.6 %) was shown with Actinomyces denticolens NCTC 11490T (Dent & Williams, 1984b). Therefore, based on sequence divergence and phylogenetic evidence, it is clear that isolates B71T and D471 represent a novel species of the genus Actinomyces.
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9 (25.33 %) predominating. This fatty acid profile was different from those of phylogenetically related species such as Actinomyces oricola (Hall et al., 2003c), Actinomyces israelii (Johnson et al., 1990) and Actinomyces bovis (Johnson et al., 1990) in which the major fatty acids are C16 : 0 (44, 38.9 and 23.7 %, respectively), C18 : 1 cis9 (37, 33.4 and 56.2 %, respectively) and C18 : 0 (14, 2.5 and 10.1 %, respectively). The fatty acid content of strain B71T demonstrated the relationship of the novel strains to the genus Actinomyces. Strain B71T contained menaquinones as the sole respiratory quinones, with MK-10 (70 %) as the major component and a low content of MK-9 (30 %).
The novel strains isolated from rumen contents could degrade xylan; a feature of their natural niche. The novel strains were readily distinguished from other species of the genus Actinomyces on the basis of phenotypic characteristics (Table 1). They differed from most of the other recognized Actinomyces species by showing strictly anaerobic growth and by having a higher optimum temperature for growth (46 °C). Other distinguishing characteristics were starch hydrolysis, sorbitol, rhamnose, cellobiose and D-arabinose fermentation and the formation of different fermentation products from glucose (Coleman et al., 1969; Collins et al., 2000; Dent & Williams, 1984a, b, 1986; Hall et al., 2005, 2003c; Hoyles et al., 2001b; Johnson et al., 1990; Nikolaitchouk et al., 2000; Pascual et al., 1999; Schaal, 1992; Stackebrandt & Charfreitag, 1990) (see Table 1). The novel strains differed from A. denticolens in their natural niche, cell morphology, nitrate reduction and DNA G+C contents. Based on the phenotypic and phylogenetic distinctiveness of the rumen isolates, a novel species, Actinomyces ruminicola sp. nov. is proposed.
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Cells are non-motile, straight rods (0.5–1.0x2.5–4.0 µm) that are Gram-positive, non-spore-forming, catalase-negative and strictly anaerobic. Colonies on PYRG agar are circular, slightly convex, white and reach approximately 0.5–0.8 mm in diameter after 48 h incubation at 46 °C. Formic acid is the main end product of glucose fermentation; acetate and lactate are also produced. Acids are produced from D-galactose, D-maltose, D-fructose (weak reaction), D-lactose, ribose, sucrose, erythritol, D-arabinose, D-xylose, cyclodextrin, rhamnose, sorbitol, cellobiose, melibiose, trehalose and melezitose. Acid production is variable from inositol, mannitol, mannose, salicin, glycogen and xylitol. Acid is not produced from glycerol, dulcitol, inulin, glucoside or adonitol. Hippurate and urea are not hydrolysed. Milk is curdled. Aesculin hydrolysis is variable. Nitrate is reduced. Methyl red test is positive, but Voges–Proskauer test is negative. The cellular fatty acids are of the straight-chain saturated and monounsaturated types, with C16 : 0, C18 : 0 and C18 : 19 predominating. The major respiratory quinones are menaquinones MK-10 (70 %) and MK-9 (30 %).
The type strain, B71T (=JCM 13352T=CGMCC 1.5030T), was isolated from cattle rumen.
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ACKNOWLEDGEMENTS |
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