Clostridium Proteoclasticum : a Ruminal Bacterium That Forms Stearic Acid from Linoleic Acid R

Clostridium proteoclasticum : a ruminal bacterium that forms stearic acid from linoleic acid R. John Wallace, Lal C. Chaudhary, Nest McKain, Neil R. McEwan, Anthony J. Richardson, Philip E. Vercoe, Nicola D. Walker & Delphine Paillard

Rowett Research Institute, Bucksburn, Aberdeen, UK

Correspondence: R.J. Wallace, Rowett Abstract Research Institute, Bucksburn, Aberdeen, AB21 9SB, UK. Tel.: 1 44 1224 716656; The aim of this study was to identify ruminal bacteria that form stearic acid (18 : 0) fax: 1 44 1224 716687; e-mail: from linoleic acid (cis-9,cis-12-18 : 2). One 18 : 0-producing isolate, P-18, isolated [email protected] from the sheep rumen was similar in morphology and metabolic properties to ‘Fusocillus’ spp. isolated many years ago. Phylogenetic analysis based on nearly full- Present addresses: Lal C. Chaudhary, length 16S rRNA gene sequence (4 1300 bp) analysis indicated that the stearate Centre of Advanced Studies in Animal producer was most closely related to Clostridium proteoclasticum B316T. Clostri- Nutrition, Indian Veterinary Research dium proteoclasticum B316T was also found to form 18 : 0, as were other bacteria Institute, Izatnagar - 243 122, India. isolated elsewhere, which occurred in the same family subclass of the low G1C% Neil R. McEwan, Institute of Rural Science, University of Wales, Llanbadarn Campus, Gram-positive bacteria, related to Butyrivibrio ﬁbrisolvens. These bacteria are not Aberystwyth, SY23 3AL, UK. clostridia, and the ability to form 18 : 0 was present in all strains in contrast to Philip E. Vercoe, Animal Science Group, proteolytic activity, which was variable. Production of 18 : 0 occurred in growing, University of Western Australia, Crawley, WA but not in stationary-phase, bacteria, which made detection of biohydrogenating 6009, Australia. activity difﬁcult, because of the inhibitory effects of linoleic acid on growth. Nicola D. Walker, l’Unite´ de Microbiologie, INRA de Clermont-Ferrand-Theix, 63122 St Gene` s Champanelle, France.

Received 2 August 2006; revised 19 September 2006; accepted 20 September 2006. First published online 18 October 2006.

DOI:10.1111/j.1574-6968.2006.00487.x

Editor: Robert Gunsalus

Keywords biohydrogenation; Butyrivibrio ; Clostridium proteoclasticum ; Fusocillus ; stearate.

description that, like the cultures themselves, has not Introduction survived. The aim of the present study was to assess the Comparison of national dietary habits reveals that the biohydrogenating activity of fresh isolates from grass-fed consumption of saturated fatty acids in dairy products is sheep and to reisolate and identify Fusocillus-like bacteria by directly correlated to the incidence of death by heart attack modern phylogenetic techniques. in different countries (Menotti et al., 1999). Saturation of fatty acids (biohydrogenation) occurs primarily in the ru- Materials and methods men (Dawson & Kemp, 1970; Viviani, 1970). Butyrivibrio fibrisolvens was identified many years ago to undertake Fresh bacterial isolates were made nonselectively immedi- biohydrogenation (Polan et al., 1964), but was observed not ately postmortem from strained ruminal digesta of four to form stearic acid (18 : 0) from linoleic acid (LA, cis-9,cis- grazing mature sheep. These were diluted in the basal 12-18 : 2). The bacteria responsible for 18 : 0 formation were medium of Bryant & Robinson (1961), plated onto petri later identified as Fusocillus spp. (Kemp et al., 1975), a genus dishes of complete Bryant & Robinson medium and

FEMS Microbiol Lett 265 (2006) 195–201 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 196 R.J. Wallace et al. incubated at 39 1C for 48 h. Colonies from 104 to 108 polysaccharide and poly-b-hydroxybutyrate were carried dilutions were selected on the basis of differing colony out as described in the ASM technical manual (Hanson & morphology and were maintained in the liquid form of M2 Phillips, 1981). G1C% content was measured by the medium (Hobson, 1969). In all growth experiments, culture Deutsche Sammlung von Mikroorganismen und Zellcultu- tubes were 12.5 1.6 cm, closed with screw caps fitted with ren GmbH (Braunschweig, Germany) using reverse-phase butyl rubber septa (Bellco Biotechnology, Vineland, NJ). All HPLC (Mesbah & Whitman, 1989). In order to determine transfers were carried out under O2-free CO2. Incubations the influence of growth phase on fatty acid metabolism, LA were at 39 1C. Inoculum volumes were 5% (v/v) of a fresh was added to the medium, to a final concentration of culture. 50 mgmL1, before inoculation, or when the OD had Bacterial strains other than P-18 were obtained from the reached 0.3 (mid-exponential phase), or when the stationary Rowett collection (SH1, SH13 and JW11), purchased from phase was reached. In each case, cultures were incubated for culture collections (DSM or ATCC) or gifted by interna- 48 h after adding LA, and then cultures were freeze-dried tional colleagues. Most cultures were obtained from and analysed for fatty acids. Electron microscopy was J. Kopecˇny´ and they are described in Kopecˇny´ et al. (2003). carried out on bacteria taken from a colony on M2 agar Clostridium proteoclasticum ‘C-proteo’ was a gift from medium, the cultures being stained using ammonium G.T. Attwood and is among the strains described by molybdate as described previously (Wallace et al., 2003). Attwood et al. (1996). Strain Su6 was a gift from K.N. Joblin Bacterial DNA was extracted using the Wizard Genomic and is described in van de Vossenberg & Joblin (2003). DNA purification kit (Promega). A full sequence of 16S In order to determine whether bacteria produced 18 : 0 rRNA gene was amplified using the universal primers P0124 from LA or a-linolenic acid (LNA, cis-9,cis-12,cis-15-18 : 3), 50-CACGGATCCGGACGGGTGAGTAACACG-30 and C1400 the isolates were inoculated into M2 liquid medium con- 50-TGGTGTGACGGGCGGTGTGT-30 in a mixture contain- taining 50 mgLAorLNAmL1 and incubated for 48 h. ing 1 mM of each of the primers, 200 mM of each of the

Cultures were then extracted and analysed for free fatty acid dNTPs, 50 mM KCl and 1.5 M MgCl2 in 10 mM Tris HCl concentrations. Where cultures failed to grow but grew on pH 8.3 buffer. 0.1 mg of DNA and 2.5 U of Taq DNA medium with no added LA, the culture was inoculated into polymerase were added to 50 mL of this mixture and medium containing 20 mgLAmL1 and the process was amplification was carried out using 35 cycles at 94 1C for repeated. Extraction and derivatization of fatty acids were 1 min followed by 55 1C for 2 min and 72 1C for 2 min. carried out by procedures based on those described by Amplification was confirmed on an agarose gel. Amplified Christie (2003) and modified by Wa˛sowska et al. (2006), DNA was cloned into the PCRs2.1-TOPO vector (Invitro- using samples (1 mL) of fresh or more usually resuspended, gen BV, Leek, the Netherlands) following the manufacturer’s freeze-dried cultures. Proteolytic activity was measured in instructions. Plasmids were isolated from recombinant triplicate 6-h incubations of stationary-phase cultures with colonies using the Wizard Plus SV miniprep kit (Promega), [14C]-labelled casein (Wallace & Brammall, 1985). and the nucleotide sequence of the insert was determined Isolate P-18 was characterized by conventional methods using an ABI PrismTM 377XL DNA sequencer. The 16S based on those of Holdeman et al. (1977). The presence of rRNA gene sequences were aligned with the CLUSTALX spores was investigated by incubating M2 cultures at 85 1C program. Distance matrices were calculated with the for 15 min, and then reinoculating fresh tubes of M2 liquid DNADIST program using the algorithm of Kimura-2 within medium. Fermentation products were determined by deri- the PHYLIP package (Felsenstein, 1989). The phylogenetic vatization and capillary gas-liquid chromatography of trees were inferred from evolutionary distances with the supernatants from cultures grown in M2 liquid with no Neighbor program of the same package. The confidence added LA (Richardson et al., 1989). Some fermentation tests level of the phylogenetic tree topology was evaluated by were carried out using API 20 A strips (bioMerieux,´ Lyon, performing 1000 bootstrap replications with the programs France) under anaerobic conditions to determine sugar SEQBOOT and CONSENSE of the same package. The P-18 utilization and indole production. Most were done by sequence used in the present work was deposited with EMBL inoculating strain P-18 into Bryant & Robinson basal under accession number AM039827. Others included strain medium with added individual sugars (5 g L1). Sugar Mz3 (AM039822), strain Mz9 (AM039823), strain CE51 utilization was considered to be positive when there was a (AM039824), strain JK612 (AM039825) and strain JK684 growth response as measured turbidimetrically at 650 nm (AM039826). using a Novaspec II spectrophotometer (Amersham Phar- macia Biotech, Little Chalfont, Bucks). Gelatin liquefaction Results and discussion was carried out as described by Holdeman et al. (1977), except that liquid M2 was the basal medium. Staining for One hundred and fifteen bacteria were isolated nonselec- cytoplasmic inclusions and chemical analysis for storage tively from individual colonies from four grazing sheep.

4 Only one strain, P-18, isolated from a 10 dilution, formed Table 1. Characteristics of stearate-producing strain P-18 in comparison 4 5 mg18:0mL1, with three others producing traces with other published stearate producers from the rumen 1 (c.2mgmL ). The growth of strain P-18 was subject to a ‘F. babra- lag period of 96 h when 50 mgLAmL1 was present in the Strain hamensis’ ‘Fusocillus’ B. hungatei Ã Ã w medium, and to shorter lag times at lower concentrations. Property P-18 P2/2 sp. T344 Su6 Isolate P-18 was a motile, strictly anaerobic Gram-negative Substrate staining bacillus with morphology indistinguishable from Arabinose 11 1 Xylose 111 1 Fusocillus babrahamensis (Fig. 1). Some of the cells were Rhamnose 11 spindle-shaped, as was described for F. babrahamensis Glucose 111 1 (Kemp et al., 1975). No spores were visible, and bacteria Fructose 111 were killed by incubation at 80 1C. Cells were motile, with a Mannose 111 single subpolar flagellum. Electron microscopy indicated Galactose 111 Sucrose 111 1 that, despite the staining reaction being negative, the cell Maltose 111 envelope morphology was Gram-positive. The concentra- Lactose 1 1 tions of fermentation products were higher from strain P-18 Cellobiose 111 1 than two Fusocillus isolates from Kemp et al. (1975), but Melibiose 111 other fermentation properties were closely similar (Table 1). Trehalose Raffinose Properties were generally similar to the stearate-producing Starch 11 strain Su6 designated by van de Vossenberg & Joblin (2003) Inulin 111 to be a Butyrivibrio hungatei. Like the Fusocillus strains, but Dextrin 11 unlike B. hungatei Su6, P-18 was xylanolytic. Conversely, Glycerol B. hungatei Su6 and strain P-18 hydrogenated LNA to 18 : 0, Erythritol Adonitol while the early Fusocillus isolates did not. Mannitol A phylogenetic tree was constructed using the 16S rRNA Dulcitol gene sequences of 15 type strains of close relatives of P-18, Sorbitol together with Butyrivibrio crossotus NCDO 2416, B. hungatei Salicin 111 Su6 (van de Vossenberg & Joblin, 2003) and Pseudobutyrivi- Aesculin 111 Inositol 1 brio ruminis JK205 (Kopecˇny´ et al., 2003), with an Escher- Lactate ichia coli-type strain as the outlier (Fig. 2). The tree divided Succinate into three main branches. The first one contained Pyruvate 11 Citrate Acetate Fumarate Pectin 111 1 Xylan 111 Cellulose Other G1C% 39.2 Glucose15 mM monensin Stearate from 1 1 a-linolenic acid Flagellum Sub-polar Sub-polar Sub-polar Gelatin liquefaction Catalase Fermentation products (mM) Butyrate 10.5 2.9 4.6 Major Propionate 0 0 0 Acetate 3.1 0.6 0.1 Major Formate 23.2 Major Lactate 7.7 0.1 0.2 Succinate 0 0

ÃProperties recorded by Kemp et al. (1975). Fig. 1. Comparative morphology of stearate-producing isolate P-18 and wFrom van de Vossenberg & Joblin (2003). the original ‘Fusocillus’ isolate. (a) Scanned from the original paper by Where there is a space rather than a number or symbol, the property was Kemp et al. (1975). (b) New isolate, strain P-18. not reported.

Ruminococcus type strains, the second contained Clostridium was identified as C. proteoclasticum based on 16S rRNA gene xylanolyticum, Butyrivibrio type strains and strains P-18 and sequence analysis (Kopecˇny´ et al., 2003). Strains P-18, Su6, JK205 and the third branch had type strains of Lachnospira, Bu43, JK205, JK669 and JK668 formed 18 : 0 from LA. The Eubacterium rectale, Roseburia and Pseudobutyrivibrio. The nearest branch contained the type strain of C. proteoclasti- sequence of P-18 was located in a cluster comprising B. cum B316T, which also formed 18 : 0, whereas bacteria from fibrisolvens D1T, B. hungatei JK615T, C. proteoclasticum elsewhere in the tree did not, including a neighbouring B316T, Su6 and JK 205, but was much closer to B316, Su6 branch containing the type strain JK615 of B. hungatei and JK205 sequences than to the others, with a high level of (Kopecˇny´ et al., 2003). All the strains producing 18 : 0 were confidence. This phylogenetic grouping and the absence of distantly related to the B. fibrisolvens type strain D1T (ATCC spores, also observed in the type strain of C. proteoclasticum 19171), the P. ruminis type strain (DSM 9787T) and the (Attwood et al., 1996) and ‘Fusocillus’ (Kemp et al., 1975), Pseudobutyrivibrio xylanivorans type strain Mz5 (DSM imply that the nomenclature of these bacteria as Clostridium 14809T; Kopecˇny´ et al., 2003). Proteolytic activity was high is probably incorrect. in several strains, including SH13 and DSM 10317, but very A more detailed positioning of different P-18-related low in most strains tested (Fig. 3). Unlike 18 : 0 production, strains was carried out by comparing its 16S rRNA gene proteolytic activity was not correlated with phylogenetic sequence with those of Butyrivibrio and related isolates position. obtained from around the world (Fig. 3). Many of the full- The influence of growth phase on 18 : 0 production by length sequences used in this study were obtained by us and P-18 was determined by adding LA to mid-exponential have been placed in sequence databases. Selected strains and stationary-phase cultures as well as LA being present from across the tree were investigated for their ability to in the medium at inoculation. When LA was added to form 18 : 0 from LA and for their proteolytic activity (Fig. stationary-phase cells, conjugated linoleic acids (CLA) 3). As before, strain P-18 clustered on a branch most closely accumulated (Table 2), predominantly as cis-9,trans- with isolates Su6 and JK205. The closely related strain JK669 11–18 : 2. No 18 : 1 or 18 : 0 was formed. Adding LA before

T 100 Ruminococcus flavefaciens ATCC 19208 Ruminococcus albus ATCC 27210T Eubacterium xylanophilum ATCC 35991T

Lachnospira pectinoschiza 150-1T Eubacterium rectale ATCC 33656T 98 T 100 Roseburia cecicola ATCC 33874 100 Roseburia intestinalis L182T 83 T 100 Pseudobutyrivibrio xylanivorans Mz5 Pseudobutyrivibrio ruminis DSM 9787T Butyrivibrio fibrisolvens D1T 86 100 Butyrivibrio hungatei JK615T

100 75 P-18 Clostridium proteoclasticum B316T

96 Su6 JK205

Butyrivibrio crossotus NCDO 2416

Clostridium xylanolyticum DSM 6555T

Escherichia coli ATCC 11775T

0.02

Fig. 2. Neighbour-joining phylogenetic tree of P-18 and related bacteria. The tree was constructed using nearly full-length 16S rRNA gene sequences (4 1300 bp). Numbers represent the signiﬁcance (percent of outcomes) of the branches (bootstrap analysis). Only numbers higher than 70 are shown. The bar indicates 2% difference in nucleotide sequence. T: Type strain.

4.3, − JK662 JK668 1.3, + Bu43 JK669 n.d., + P-18 * 1.2, + JK205 n.d., + Su6 0.8, + UC142 19.7, + JK724 C-proteo 12.7, + B316T, C. proteoclasticum, type strain H17c SA NCFB 2435 NCFB 2432 − DSM10295 0.9, JK611 n.d., − JK614 0.7, − − JK615, B. hungatei, type strain 0.9, NCFB 2398 NCFB 2434 − NCFB 2222 n.d., ATCC19171, B. fibrisolvens, type strain 1.4, − WV1 JK684 0.1, − JK730 0.7, − OB156 LP1265 JK626 − S2/10 n.d., JK23/2 Mz4 30.3, − − JK618 0.4, Mz8 n.d., − Mz9 − SH13 56.7, NCFB 2223 DSM10296 34.4, − NCFB 2397 Mz5, P. xylanivorans, type strain 53.1, − − JW11 0.6, D6/1 n.d., − pC-XS2 pC-XS7 pC-XS6 DSM9787, P. ruminis, type strain 0.4, − NCFB 2399 DSM10317 48.7, − O/10 − JK170 n.d., − SH1 17.4, − CE51 n.d., − JK663 9.5, NCFB 2249 B835 JL5 JK729 0.4, − JK612 JK10/1 0.5, − SR85 Mz7 DSM10316 n.d., − Mz6 0.4, − n.d. – not JK633 determined Mz3 0.01

Fig. 3. Proteolytic activity and stearic acid formation from individual strains in a Neighbour-joining phylogenetic tree of Butyrivibrio fibrisolvens and related bacteria. The tree was constructed using nearly full-length 16S rRNA gene sequences ( 4 1300 bp). The numbers in the tree indicate the significance (percent of outcomes) of the branches (bootstrap analysis). Only numbers higher than 60 are shown. The underlined text alongside many of the bacterial strains describes proteolytic activity and the ability to form 18 : 0 from LA. The first number is proteolytic activity in units mg 14C-casein hydrolysed h1 mg protein1, the second 18 : 0 production (1) or no 18 : 0 production ( ). Measurements were carried out on three replicate cultures.

Table 2. Influence of growth phase on the metabolism of LA by strain References P-18 Attwood GT, Reilly K & Patel BKC (1996) Clostridium 1 Products (mgmL ) proteoclasticum sp. nov., a novel proteolytic bacterium from trans- cis-9,trans- the bovine rumen. Int J Syst Bacteriol 46: 753–758. Treatment 18 : 0 11-18 : 1 11-18 : 2 Bryant MP & Robinson IM (1961) An improved nonselective LA added before inoculation 11.6 14.3 0.5 culture medium for ruminal bacteria and its use in LA added in exponential phase 11.2 12.7 0.2 determining the diurnal variation in numbers of bacteria in LA added in stationary phase 1.1 0 28.2 the rumen. J Dairy Sci 44: 1446–1456. Christie WW (2003) Lipid Analysis. Isolation, Separation, Identification and Structural Analysis of Lipids, The Oily Press, Bridgwater, England. inoculation or in the exponential phase resulted in the Dawson RMC & Kemp P (1970) Biohydrogenation of dietary fats formation of roughly similar concentrations of 18 : 1 and in ruminants. Physiology of Digestion and Metabolism in the 18 : 0, with minimal formation of CLA (Table 2). Kepler & Ruminant (Phillipson AT, ed), pp. 504–518. Oriel Press, Tove (1967) demonstrated that, in B. fibrisolvens,isomer- Newcastle upon Tyne, UK. ization of LA to CLA did not require cofactors and indeed Felsenstein J (1989) PHYLIP - Phylogeny Inference Package occurred aerobically in whole cells or cell extracts. In (Version 3.2). Cladistics 5: 164–166. contrast, the subsequent conversion of CLA to VA was Hanson RS & Phillips JA (1981) Chemical composition. Manual NADH-dependent (Hunter et al., 1976). Thus, it seems of Methods for General Bacteriology (Gerhardt P, Murray RGE, probable that the absence of 18 : 0 formation in stationary- Costilow RN, Nester EW, Wood WA, Krieg NR & Briggs phase C. proteoclasticum wascausedbyalimitedsupplyof Phillips G, eds), pp. 328–362, Washington, DC. reducing equivalents once the energy sources in the Hobson PN (1969) Rumen bacteria. Methods in Microbiology, Vol. medium had been depleted. 3B. (Norris JR & Ribbons DW, eds), pp. 133–139. Academic The growth-phase results also mean that, in order to Press, London. demonstrate the formation of 18 : 0 by P-18, it is necessary Holdeman LV, Cato EP & Moore WEC (1977) Anaerobe to grow the bacteria in the presence of LA. Given the growth Laboratory Manual. Blacksburg, Virginia 24060: V.P.I. inhibition that occurs when LA is added to the medium, Anaerobe Laboratory, Virginia Polytechnic Institute and State 18 : 0 formation by P-18 can sometimes be difficult to University. demonstrate. In our initial studies, which preceded the Hunter WJ, Baker FC, Rosenfeld IS, Keyser JB & Tove SB (1976) experiments reported here, we were unsuccessful in isolating Biohydrogenation of unsaturated fatty acids. Hydrogenation stearic acid-forming bacteria. The reasons for this are now by cell-free preparations of Butyrivibrio fibrisolvens. J Biol obvious, namely that bacteria must be growing in order to Chem 251: 2241–2247. carry out biohydrogenation; yet, the unsaturated fatty acids Kemp P, White RW & Lander DJ (1975) The hydrogenation of themselves have a strong tendency to inhibit growth. These unsaturated fatty acids by five bacterial isolates from the sheep properties undoubtedly must have hampered the isolation rumen, including a new species. J Gen Microbiol 90: 100–114. of more stearate producers by ourselves and others. Kepler CR & Tove SB (1967) Biohydrogenation of unsaturated fatty acids. 3. Purification and properties of a linoleate D12-cis, D11-trans-isomerase from Butyrivibrio fibrisolvens. J Biol Chem Acknowledgements 242: 5686–5692. Kopecˇny´ J, Zorec M, Mrazek´ J, Kobayashi Y & Marinsˇek-Logar R The Rowett Research Institute receives funding from the (2003) Butyrivibrio hungatei sp. nov. and Pseudobutyrivibrio Scottish Executive Environmental and Rural Affairs Depart- xylanivorans sp. nov., butyrate-producing bacteria from the ment. Our thanks are due to Florian Pizette, Mariette rumen. Int J Syst Evol Microbiol 53: 201–209. Barbier, Gemma Laughton and Julie Charbonnier, who Menotti A, Kromhout D, Blackburn H, Fidanza F, Buzina R & initiated some of the experiments. L.C.C. was in receipt of a Nissinen A (1999) Food intake patterns and 25-year mortality Wellcome Travelling Fellowship. We thank David Brown and from coronary heart disease: cross-cultural correlations in the Maureen Annand for their technical help and expertise and Seven Countries Study. The Seven Countries Study Research Tim King for electron microscopy. D.P. was funded by the Group. Eur J Epidemiol 15: 507–515. EC Framework 6 Integrated Project, ‘LIPGENE’. We record Mesbah M & Whitman WB (1989) Measurement of with gratitude the generosity of Jan Kopecˇny´, Graeme Att- deoxyguanosine/thymidine ratios in complex mixtures by wood and Keith Joblin in providing bacterial strains and high-performance liquid chromatography for determination thank them for their invaluable advice in correspondence of the mole percentage guanine1cytosine of DNA. J and discussions. Chromatog 479: 297–306.

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