Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Correspondence
R. John Wallace
rjw{at}rri.sari.ac.uk
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ABSTRACT |
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Present address: Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido, Japan.
Present address: AgResearch, Palmerston North, New Zealand.
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INTRODUCTION |
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E. pyruvativorans was named as such because it grew most rapidly on pyruvate (Wallace et al., 2003). Since pyruvate is not abundant extracellularly in the rumen (Wallace, 1978
), it appeared that the true substrate for its growth in vivo was probably amino acids, although the net utilization of C3 and C4 volatile fatty acids (VFAs) and a slight utilization of lactate suggested that E. pyruvativorans might have a wider metabolic role. The present study was undertaken to investigate the metabolic properties of E. pyruvativorans in greater detail, particularly the mechanisms of VFA and lactate utilization. Its properties illustrate a metabolic strategy analogous to that used by Clostridium kluyveri and possibly other non-saccharolytic bacteria, which enables these bacteria to cope with conditions of transient availability of energy sources under anaerobic conditions.
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METHODS |
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C sources for growth of E. pyruvativorans.
Growth experiments to determine the range of substrates that could be fermented by E. pyruvativorans were carried out by addition of the substrates at 5 g l1 to M2 basal medium, which is the liquid form of the ruminal-fluid-containing medium of Hobson (1969) with no added carbohydrate or lactate, or to CRT medium to which was added 10 mM sodium acetate, sodium propionate and sodium butyrate (CRT+VFA medium). Sodium oxaloacetate was added as a filter-sterilized solution after autoclaving, as it is broken down during autoclaving, releasing pyruvate. The inoculum for the former set of media was grown in a fresh liquid M2 culture, and, for the latter, in CRT+VFA medium. Growth (OD650) was determined hourly in a Novaspec II spectrophotometer (Amersham Pharmacia Biotech).
Growth on lactic acid.
One approach used to try to enhance growth of E. pyruvativorans on lactate was co-culture with the H2/CO2-utilizing methanogenic archaeon Methanobrevibacter smithii. M. smithii strain PS (ATCC 35061) was grown for 11 days in 10 ml M2 basal medium, either with no addition or with added 5 g l1 sodium DL-lactate or sodium pyruvate under an atmosphere of 80 % H2/20 % CO2. E. pyruvativorans was grown overnight on liquid M2 medium and 0·5 ml was inoculated into the 11 day culture of M. smithii. Growth was followed turbidimetrically as before. After stationary phase was reached, samples of the headspace gas were analysed for H2 and CH4 by GLC (Stewart & Richardson, 1989).
The influence of the gas phase was investigated further, using anaerobic chambers with different gas phases. One was filled with O2-free CO2, the other with a mixture of 80 % N2, 10 % CO2 and 10 % H2. Three different media were used: M2 basal medium, M2 basal medium with added 5 g sodium pyruvate l1, and M2 basal medium with added 5 g sodium DL-lactate l1. Nine tubes of each medium were inoculated from a fresh M2 culture into culture tubes with fixed screw-caps. Three were incubated at 39 °C as usual, and three were transferred to each anaerobic chamber, where their caps were replaced by sterile cotton-wool bungs. Growth was followed turbidimetrically as before at 3 h intervals. Screw-caps were replaced before the tubes were removed from the chambers, and exchanged with the cotton wool bungs when the tubes were returned to the chambers.
In order to assess if lactate utilization might be coupled to the utilization of a more oxidized substrate, E. pyruvativorans was inoculated into medium containing 5 g sodium DL-lactate l1 and 1, 3 or 5 g sodium aspartate l1. Growth was followed turbidimetrically as before.
The utilization of different racemers of lactate was investigated using M2 basal medium with added 5 g sodium DL-lactate l1. Cultures were inoculated from a fresh culture grown on M2 basal medium with added 5 g sodium pyruvate l1. When cultures reached stationary phase, they were centrifuged (15 000 g, 15 min, 4 °C) and the supernatant fluid was stored at 20 °C until it was analysed for lactate and VFAs.
The fate of lactate-C was determined using L-[14C]lactate. The inoculum was a fresh culture grown on M2 basal medium. The growth medium was M2 basal medium with added 1 g sodium pyruvate l1 and 0·2 µCi (7·3 kBq) sodium L-[U-14C]lactate (5·99 GBq mmol1; Amersham Biosciences). Growth was followed turbidimetrically. When stationary phase was reached, a sample of the whole culture was taken for radioactivity measurement by liquid scintillation spectrometry and the remaining culture was centrifuged (15 000 g, 15 min, 4 °C). The radioactivity in a sample from the supernatant was measured, and a further sample was separated by HPLC (see below) and 0·5 ml fractions of eluent were measured for radioactivity. The radioactivity associated with fractions corresponding to the peaks of each VFA and lactate was summed. The pellet was washed once in 0·1 M sodium phosphate buffer, pH 7·0, and samples were taken for measurement of 14C radioactivity.
Lactate dehydrogenase (LDH) was measured in sonicated cells of E. pyruvativorans and Selenomonas ruminantium Z108, an isolate obtained from a sheep at the Rowett Research Institute and maintained in the Institute's culture collection. Both bacteria were grown overnight in the liquid form of complete M2 medium, which contains sodium lactate, or in M2 basal medium (no lactate or sugars) with added sodium pyruvate (5 g l1). The cultures were centrifuged, sonicated and LDH was measured in cell-free extracts by the method described previously (Wallace, 1978) using NADH and pyruvate as substrates.
Amino acid metabolism.
In order to determine its N requirements, E. pyruvativorans was subcultured by inoculating 5 % of a fresh M2 culture into Chen & Russell (1988) basal medium, to which was added various combinations of 5 g sodium pyruvate l1 and N sources. The added N sources were 15 g PCH l1, 15 g l1 of a mixture of 20 amino acids (Atasoglu et al., 1998
) and 3 g ammonium chloride l1. After incubating for 24 h, the cultures were passaged again using a 5 % inoculum, and growth of the culture was assessed by measuring OD650.
In order to determine if single amino acids could replace PCH as the growth substrate, each of the 20 L-amino acids found in bacterial proteins was added individually to CR medium (CRT with no added PCH) at a concentration of 15 g l1, along with 10 mM sodium propionate. Growth was followed turbidimetrically as before.
Amino acid utilization was determined in M2 basal medium (no added carbohydrate or lactate) with added 10 g l1 algal protein hydrolysate (APH) labelled with 13C. The APH added to the medium consisted of 20 % (w/w) Celtone C (99 % atom % 13C) and 80 % (w/w) Celtone U (unlabelled). The APH preparations, consisting mainly of small peptides and amino acids, were obtained from Celtone, Martek Biosciences. APH-containing medium formed a precipitate during autoclaving, which was removed by centrifugation (5000 g, 10 min). The inoculum was an overnight culture of E. pyruvativorans grown in the same medium. A 5 % (v/v) inoculation was made into triplicate tubes, the tubes were incubated at 39 °C, and optical density was followed turbidimetrically. Immediately stationary phase was reached, the cultures were centrifuged (15 000 g, 15 min, 4 °C). The supernatant was stored at 20 °C. Subsequently, the supernatants were thawed for analysis of VFAs, amino acid concentrations and isotopic enrichment. The pellet was washed once and also stored frozen before analysis for protein and amino acids.
The influence on growth of amino acids whose catabolism is likely to give rise to pyruvate was determined by adding 1 g l1 each of alanine, glycine, serine and threonine to M2 basal medium or CRT medium. Growth was determined turbimetrically in four replicate cultures.
VFA metabolism.
The influence of acetate, propionate and butyrate on growth was determined by adding 10 mM sodium acetate, 10 mM sodium propionate or 10 mM sodium butyrate to CRT medium. An additional medium (CRT+VFA) contained added 10 mM each of the VFAs sodium acetate, sodium propionate and sodium butyrate. Growth was followed turbidimetrically as before. When stationary phase was reached, the cultures were centrifuged (15 000 g, 15 min, 4 °C), and fermentation products were determined as described below.
In order to investigate the use of exogenous acetate in the formation of higher VFAs, bacteria were inoculated into CRT+VFA medium containing 2400 Bq ml1 of sodium [1-14C]acetate (2·07 GBq mmol1; Amersham Biosciences ). After 48 h incubation, cells were removed by centrifugation, and radioactivity was determined in the supernatant fluid by liquid scintillation spectrometry. Portions (0·05 ml) of the supernatants were analysed for VFAs by HPLC (Rooke et al., 1990). Fractions corresponding to valeric acid and caproic acid were collected and analysed by liquid scintillation spectrometry.
Analyses.
Protein was determined using the Folin reagent after alkaline digestion (Herbert et al., 1971). VFAs and DL-lactate were analysed routinely by GLC after derivatization (Richardson et al., 1989
). L-Lactate was determined enzymically (Goodall & Byers, 1978
). Amino acid concentrations were measured by HPLC of phenylisothiocyanate derivatives (Bidlingmeyer et al., 1984
) after HCl hydrolysis (Wallace & McKain, 1990
). Enrichment of 13C in amino acids was measured by GC-MS analysis of tertiary butyl dimethylsilyl (TBDS) derivatives (Atasoglu et al., 1998
). Enrichment in VFAs was determined in a similar way by GC-MS. The fatty acids were derivatized and separated as before. The GC column was connected directly to the ion source of a Thermo Finnigan TRIO-1 mass spectrometer. The mass spectrometer was operated under electron impact conditions with the following source parameters: electron energy 70 eV, emission current 150 µA, and source temperature 200 °C. Selective ion recording was used to monitor the M57 (massTBDS) and M57+n ions of interest, with a dwell time of 30 ms on each ion. For acetate, propionate, isobutyrate, butyrate, isovalerate, valerate and caproate, the M-57 ions monitored were m/z 117, 131, 145, 145, 159, 159 and 173 respectively. Deconvolution analysis, whereby the relative concentration of all isotopomers is determined (rather than only Mr+n) was carried out by mathematical procedures described by Campbell (1974)
.
Statistical analysis.
The results are, except where stated otherwise, means obtained from triplicate cultures. Statistical significance of differences between means (SED) was determined by Student's t-test. Coefficients of variation were pooled between treatment groups, as variance in the different treatment groups was similar.
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RESULTS |
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The use of total lactate, determined by GC, and that of the L-isomer, determined enzymically, was measured after growth on M2 basal medium with added 5 g sodium DL-lactate l1. The total concentration of lactate fell from 38·8 to 31·7 (SD 2·6) mM, a decrease of 7·1 mM. L-lactate fell from 22·4 to 17·7 (SD 0·2) mM, a decrease of 4·7 mM. Thus, the main racemer used was L-lactate. L-[14C]lactate was used to determine the fate of the lactate that was metabolized, using the same medium to which a lower concentration of lactate was added (Table 4). Lactate concentration fell by 43 %, whereas the radioactivity in the lactate peak fell by 55 %, again consistent with greater utilization of the L-racemer. Less than 1 % of the 14C was incorporated into cell material. The main product was acetate (48 %), with smaller quantities reaching propionate, butyrate, valerate and caproate (1, 9, 4 and 10 % respectively). Since the main products formed during growth were valerate and caproate, it was clear that most valerate-C and caproate-C originated elsewhere.
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VFA metabolism
Growth in the ruminal-fluid-containing general-purpose medium M2 resulted in a decrease in propionate concentration, little change in acetate or butyrate concentration, and increases in valerate and caproate concentrations (Table 5). Growth on CRT medium was much less dense, with lower concentrations of VFA formed, which were distributed among the individual acids. When sodium pyruvate was added to CRT medium, VFA production increased markedly (Table 5
), indicating a ratio of products from pyruvate (by subtracting the concentration of acid produced in the absence of pyruvate) of 10 acetate : 3 butyrate : 6 caproate : 3 lactate. Adding any one of acetate, propionate or butyrate, or a mixture of the three, to CRT medium enhanced the growth rate of E. pyruvativorans (Fig. 2
) without having a major effect on the final bacterial protein concentration. Adding acetate resulted in increased concentrations of all VFAs, while adding propionate and butyrate resulted in the formation of valerate and caproate, respectively (Table 5
).
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DISCUSSION |
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Role of E. pyruvativorans as a ruminal HAP species: amino acid metabolism and growth requirements
E. pyruvativorans grew poorly on amino acids in comparison with PCH, indicating a requirement for peptides rather than single amino acids. The original HAP species isolated by Russell and his colleagues fermented a limited range of amino acids, and growth of some was better on free amino acids, while growth of others benefited from peptides (Russell et al., 1991). Although peptides stimulate the growth of certain ruminal bacteria, an absolute requirement for peptides rather than amino acids is not common; it may indicate that some amino acid transport systems may be absent from E. pyruvativorans and other HAP species.
The amino acids metabolized to the greatest extent by E. pyruvativorans were alanine, followed by leucine, serine and proline, although most amino acids were used to some extent. The concentrations of lysine, histidine and tyrosine changed least. No single amino acid supported growth. The possibility that amino acids were metabolized in coupled Stickland-like reactions was not investigated because of the requirement of the bacterium for peptides. When alanine, glycine, serine and threonine were added to M2 basal medium, the cell density increased to 0·373 g protein l1, similar to the final cell density with pyruvate as the growth substrate (0·399 g protein l1; Table 3). A puzzling observation was that none of the amino acids was exhausted during growth on PCH, which prompts the question, also posed below for lactate utilization what other limitation caused growth to cease?
Utilization of lactate and other compounds for growth
The ability of E. pyruvativorans to grow rapidly on pyruvate presumably reflects only the central role of pyruvate in metabolism. A similar situation may apply to the utilization of crotonate and vinyl acetate. Lactate is different in that it is a common product of ruminal bacteria (including E. pyruvativorans during growth on pyruvate) that will always be present in ruminal digesta to some extent, and which can accumulate under certain dietary conditions. An earlier study (Wallace et al., 2003) indicated that lactate appeared to stimulate growth of E. pyruvativorans to a slight extent.
One possibility for the incomplete utilization of lactate previously observed is that E. pyruvativorans ferments lactate, but that the extent of this activity is masked in pure culture because H2 accumulates in the closed culture tube. This would be analogous to the interspecies H2 transfer which enables consortia of other micro-organisms to ferment compounds unable to be fermented by single species because of the accumulation of H2 (Wolin et al., 1997). Growth was stimulated to a small extent by lactate, and there was no doubt that lactate was metabolized, but growth on lactate was not stimulated in co-culture with a H2-utilizing species, or by growth in open tubes in a CO2 gas phase. Since lactate is more reduced than pyruvate, growth on lactate may introduce problems in regenerating oxidized cofactors (see below). Virtually no lactate was incorporated when [14C]lactate was added to medium containing PCH, although some [14C]lactate was converted to other fermentation products. No amino acid was exhausted from the medium, and addition of aspartate gave no stimulation, indicating that the limitation was not caused by the availability of amino acids. The LDH activity of E. pyruvativorans was much less than that of S. ruminantium, which is a major species that utilizes lactate as a growth substrate in the rumen. Furthermore, lactate was formed during growth on pyruvate. Therefore, a low, reversible LDH activity may explain the slow growth on lactate. The factor that limits more complete lactate utilization remains unclear, however, but presumably, as lactate only accumulates above trace concentrations in the rumen when digestive disturbances occur, lactate utilization may be only an energy-scavenging activity of E. pyruvativorans. Lactate utilization was observed in 2 of 14 HAP isolates studied by Attwood et al. (1998)
. Neither of these was a Clostridium/Eubacterium species. None of the isolates of Russell and colleagues (Chen & Russell, 1989
; Russell et al., 1991
) fermented lactate.
Use of VFAs during growth
Ruminal bacteria typically produce short-chain VFAs, particularly one or more of acetate, propionate and butyrate, during growth. In contrast, in cultures of E. pyruvativorans, the concentrations of propionate and butyrate appeared to decline as valerate and caproate were formed (Wallace et al., 2003). The present study demonstrates that the shorter VFAs are required for optimal growth rate on amino acids but not on pyruvate, and that the mechanism of elongation resembles reverse
-oxidation of fatty acids. The origin of the C2 groups appears to be acetyl CoA, generated from amino acids but not from exogenous acetate. A likely metabolic scheme is presented in Fig. 3
. This has close similarities to the scheme proposed by Smith et al. (1985)
for C. kluyveri. The scheme explains why metabolism leads to only two C atoms of butyrate and caproate being labelled in medium in which amino acids are the source of label (Table 7
). In contrast, isobutyrate and isovalerate are formed directly from amino acids, resulting in a fully labelled C-skeleton. With valerate, roughly equal concentrations of 2-C and 3-C label were found in the product, for reasons that are not clear. The enzymic mechanism for this fatty acid elongation is unknown, although it seems analogous to reverse
-oxidation. E. pyruvativorans has a gene with high sequence similarity (>50 % identity in a 616 bp fragment of E. pyruvativorans DNA) to bacterial 3-hydroxybutyryl-CoA dehydrogenases from Fusobacterium nucleatum and Clostridium acetobutylicum, and >40 % identity to 3-hydroxyacyl-CoA dehydrogenase from eukaryotes, including man (P. Louis, personal communication). Madan et al. (1973)
purified an NADP-linked 3-hydroxybutyrate dehydrogenase from C. kluyveri, which had a narrow substrate specificity to 3-hydroxybutyrate. Madan et al. (1973)
speculated that a particulate NAD-linked activity from the same species might have broader specificity.
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Ecological niche of E. pyruvativorans
HAP bacteria were first isolated from the rumen by Russell and his colleagues (Chen & Russell, 1988, 1989
; Russell et al., 1991
; Paster et al., 1993
) and subsequently by Attwood et al. (1998)
, McSweeney et al. (1999)
and Eschenlauer et al. (2002)
. They were isolated by their ability to grow on PCH in the absence of sugars. E. pyruvativorans was isolated in the same way and found to have many physiological characteristics in common with other HAP isolates (Eschenlauer et al., 2002
). Notably, E. pyruvativorans was non-saccharolytic, sensitive to monensin, and formed valerate and/or caproate. Other groups of ruminal HAP bacteria may therefore have a similar physiology to E. pyruvativorans. Whether amino acid utilization is their main source of energy is unclear. E. pyruvativorans grows rapidly on pyruvate, in contrast to the related species and other HAP bacteria, which are reported to ferment pyruvate only weakly (Attwood et al., 1998
) or not at all (Russell et al., 1988
; Chen & Russell, 1989
). However, pyruvate is not present in high concentrations in ruminal fluid (Wallace, 1978
). The partial utilization of lactate and other metabolites suggests that the niche which E. pyruvativorans occupies may be one of a scavenger, converting amino acids and other compounds to pyruvate in order to conserve energy via the oxidation of pyruvate to acetate and the generation of ATP. There must presumably be some benefit in obligately non-saccharolytic species having a non-saccharolytic physiology in an environment in which sugars are available transiently. The selective advantage in not being able to use sugars is not clear, however. It may simply be that the problem of H2 disposal in these species is exacerbated when sugars are fermented. Establishing the presence or otherwise of the enzymes of the EmbdenMeyerhofParnas pathway and sugar transport systems would help establish why sugars are not fermented.
Parallels with other bacteria
Phylogenetic analysis based on 16S rDNA similarity (Wallace et al., 2003) indicated that E. pyruvativorans is most closely related (92 % identity) to an non-saccharolytic Eubacterium (strain C2) isolated from the rumen (Attwood et al., 1998
), but is also related to non-saccharolytic, protein-fermenting oral bacteria, including Eubacterium timidum (Cheeseman et al., 1996
), Eubacterium minutum (tardum) (Wade et al., 1999a
, b
) and Eubacterium brachy (Hamid et al., 1994
), with identities of 89, 89 and 88 % respectively. The bacteria share a non-saccharolytic physiology as well as a common location in the digestive tract, suggesting a common progenitor. The ability of bacteria like E. pyruvativorans to scavenge energy effectively from amino acids and short-chain fatty acids may provide an ecological advantage in the oral cavity. One can imagine that periodontal bacteria would always have an ample supply of protein and amino acids, as would HAP bacteria in the rumen, where ciliate protozoa break down bacterial protein rapidly (Williams & Coleman, 1997
). The large intestine may prove to be a similarly suitable ecosystem for non-saccharolytic bacteria like E. pyruvativorans.
The parallels have already been mentioned between E. pyruvativorans and the soil anaerobe C. kluyveri in their use of acetate, propionate or butyrate to enable growth on other substrates (Bornstein & Barker, 1948; Bartsch & Barker, 1961
). Another similarity is the utilization of vinyl acetate and crotonate (Bartsch & Barker, 1961
; Madan et al., 1973
; Söhling & Gottschalk, 1996
; Wolff et al., 1993
). Neither species ferments common sugars. C. kluyveri ferments ethanol to caproate in the presence of acetate, propionate or butyrate, while E. pyruvativorans, although it does not use ethanol, uses amino acids and lactate, via pyruvate, to support growth, which is stimulated by acetate, propionate or butyrate. Caproate and/or valerate are formed depending on the composition of the medium by both species. n-Heptanoate is not formed by either species. Unlike C. kluyveri (Bartsch & Barker, 1961
; Madan et al., 1973
; Söhling & Gottschalk, 1996
; Wolff et al., 1993
), growth of E. pyruvativorans is not stimulated by succinate. E. pyruvativorans grows on pyruvate, while C. kluyveri does not (Bornstein & Barker, 1948
). Thus, although there are clear biochemical differences between E. pyruvativorans and C. kluyveri, there appears to be a common strategy. Non-saccharolytic bacteria with mechanisms of energy conservation used by C. kluyveri may be more widespread than is generally recognized.
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ACKNOWLEDGEMENTS |
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Received 25 March 2004;
revised 25 May 2004;
accepted 9 June 2004.
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