Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 05 Jan 2019 20:13:09 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, zyxwvutsr July 1996, p. 753-758 Copyright zyxwvutsrqpo 0 1996, International Union of Microbiological Societies 0020-771 3/96/$04.00 zyxwvutsrqp + 0 Vol. 46, No. 3 zy Clostridium proteoclasticum sp. nov., a Novel Proteolytic Bacterium from the Bovine Rumen G. T. ATIWOOD,'* K. REILLY,' AND B. K. C. PATEL' AgResearch, Grasslands Research Centre, Palmerston North, New Zealand, ' and Faculty of Science and Technoloa, Grifith University, Nathan, Brisbane, Queensland, Australia, 41 I1 A novel proteolytic bacterium was isolated from rumen contents of New Zealand cattle grazing fresh forage and was designated strain B316T (T = type strain). Strain B316T cells were straight to slightly curved rods (width, 0.4 to 0.6 pm; length, 1.3 to 3.0 pm) that were gram-positive and possessed a single subterminal flagellum. This isolate did not produce catalase, indole, ammonia, lipase, or lecithinase or reduce nitrate, but it did produce a curd reaction with milk. Strain B316Twas proteolytic, hydrolyzing casein and fraction I leaf protein. The crude proteinase was predominantly the serine type, but some cysteine proteinase and metallo- proteinase activities were also detected. The DNA base composition of strain B316Twas 28 mol% G+C. A 16s ribosomal DNA sequence analysis of strain B316T indicated that it was most closely related to a member of clostridial cluster XIVa, viz., zyxwvutsrq Clostridium aminophilum, an amino acid-fermenting organism isolated from the rumen; the similarity value was 92.2%. The results of the phenotypic characterization analysis, G+C content analysis, and phylogenetic analysis of the 16s ribosomal DNA sequence set strain B3MT apart from all of the members of cluster m a. We propose that strain B316T should be designated a new species of the genus Clostridium, Clostridiumproteochticum. Strain B316 is the type strain and has been deposited in the American Type Culture Collection as strain ATCC 51982. In New Zealand ruminants, the breakdown of plant protein in the rumen can lead to the loss of up to 50% of the available protein (14) and therefore to the inefficient use of pasture nitrogen. Bacteria are the most active proteolytic organisms in the rumen (2, 18), and their populations are influenced by the diet of the animal and the physical form of the feed. Many rumen bacteria are able to degrade protein, and members of the genera Prevotella, Ruminobacter, Selenomonas, Butyn'vibrio, and Streptococcus are commonly identified as being proteo- lytic. More recently, members of the genera Peptostreptococcus and Clostridium have been identified as being important in peptide and amino acid fermentation in the rumen (6, 7, 20). In a recent study of the proteolytic bacteria present in New Zealand cattle, species belonging to the genera Streptococcus, Eubacterium, and Butyrivibrio were identified as important members of the proteolytic flora (1). One highly proteolytic strain, strain B3MT (T = type strain), was initially identified as a Butyrivibrio-like organism. However, further phenotypic characterization and a phylogenetic analysis of the 16s ribo- somal DNA (rDNA) sequence of this strain identified it as a member of a new species of the genus Clostridium. We propose that this organism should be named Clostridiurn proteoclasti- cum sp. nov. MATERLALS AND METHODS Isolation and characterization of strain zyxwvutsrq B316T. Strain B316T was isolated from a rumen sample from a cow grazing in a fresh pasture as described previously (1). Unless otherwise indicated, all procedures were carried out anaerobically in Hungate tubes under a CO, atmosphere or in an anaerobic chamber containing a 92% CO,-8% H2 atmosphere. Substrate utilization tests were performed with CC medium (13), and the substrates tested were added at the following final concentrations: adonitol, 0.5% (wthrol); arnygdalin, 0.5% (wthol); arabinose, 0.5% (wt/vol); cellobiose, 1.0% (wt/vol); cellulose, 1.0% (wthol); dextrin, 1.0% (wthol); dulcitol, 1.0% (wthol); erythritol, 0.5% (wthol); fructose, 1.0% (wtivol); galacturonic acid, 1.0% (wt/vol); galactose, 1.0% (wt/vol); glucose, 1.0% (wtivol); * Corresponding author. Mailing address: AgResearch, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand. Phone: 64 6 356 8019. Fax: 64 6 351 8003. Electronic mail address: attwoodg@agresearch.cri.nz. glycerol, 1.0% (wthol); glycogen, 0.5% (wthol); inositol, 1.0% (wtivol); inulin, 1.0% (wtivol); lactate, 0.77% (wtivol); lactose, 1 .O% (wtivol): maltose, 1.0% (wtivol); mannitol, 1.0% (wtivol); mannose, 1.0% (wtivol); melezitose, 0.5% (wt/vol); melibiose, 0.5% (wtivol); rhamnose, 1.0% (wtivol); salicin, 1.0% (wti vol); sorbitol, 1 .O% (wtivol); sorbosc, 1.0% (wt/vol); starch, 1.0% (wtivol); su- crose, 1.0% (wtivol); trehalose, 0.5% (wt/vol); xylan, 1.0% (wtivol); and xylosc, 1.0% (wtivol);. Routine growth of strain B316" was carried out on CC-glucose medium, which is CC medium supplemented with 1.0% glucose. Blood agar plates for hemolysis tests were prepared from blood agar base supplementcd with 5% horse blood (Life Technologies, Auckland, New Zealand). End prod- ucts of fermentation were analyzed by high-performance liquid chromatography (HPLC). Samples (1 ml) of culture were centrifuged at 12,000 X g for 10 min at 4"C, and 50 p.1 of the supernatant was analyzed by using an Aminex type HPX 87H ion exclusion column attached to a Bio-Rad isocratic HPLC system; 5 mM H,SO, was used as the eluant. Peaks were detected with a differential refrac- tometer, identified by comparison with standards, and integrated by using a Bio-Rad model 700 chromatography workstation (version 3.71). Gas chromatog- raphy (26) and growth stimulation and biochemical tests (1 1) were carricd out as previously described. Formation of endospores was investigated by growing cells on chopped meat agar slants (Oxoid, Basingstoke, United Kingdom) and staining for spores by the Schaeffer-Fulton method (9) and by the heat test (11). The G+C content was estimated as previously described (19). Proteinase assays and inhibitors. Proteinase assays were carried out as pre- viously described (1) by using casein and fraction I leaf protein (FILP) labelled with fluorescein (25). Inhibition assays were carried out by using the azocasein assay (5) and including inhibitors (Sigma Chemical Co., St. Louis, Mo.) at the following final concentrations: phenylmethylsulfonyl fluoride, 3 mM; N-tosyl-l- lysine chloromethyl ketone, 1 mM; N-tosyl-1-phenylalanine chloromethyl ke- tone, 1 mM; EDTA, 10 mM; merthiolate, 5 mM; p-chloromcrcuribenzoate, 2.5 mM; o-phenanthroline, 2.5 mM; and pepstatin A, 200 pg rn1-l. Electron microscopy. Electron microscopy of negatively stained cells and of thin sections was carried out with a Philips model 201C electron microscope. Whole-cell preparations were negatively stained with 10h phosphotungstic acid and mounted on Formvar-coated copper grids. Thin sections were prepared from bacterial cell pellets as previously described (24). rDNA analysis. DNA was extracted (21), and the ribosomal 16s gene was amplified by using the following universal primers in a PCR: primers fdl (CCG AAT TCG TCG ACA ACA GAG 7TT GAT CCT GGC TCA G) and rdl (CCC GGG ATC CAA GCT TAA GGA GGT GAT CCA GCC). The PCR product was purified by using GlassMAX DNA isolation spin cartridges and digested with EcoRI and BamHI as recommended by the manufacturer (Gibco BRL, Life Technologies, Auckland, New Zealand). Because of an internal EcoRI site within the B316T 16s rRNA gene, two fragments resulted, and each fragmcnt was ligated separately into appropriately digested pUC19. The ligated DNAs were used to transform competent Eschen'chia coli DtISa, and transformants containing 16s rRNA gene fragments were detected by blue-white color selec- tion and verified by plasmid minipreparations (15). The 16s clones were se- quenced (23) with a model 373A automated sequencer (Applied Biosystems, 753