The proteolytic systems of ruminal microorganisms Rj Wallace

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Rj Wallace. The proteolytic systems of ruminal microorganisms. Annales de zootechnie, INRA/EDP Sciences, 1996, 45 (Suppl1), pp.301-308. ￿hal-00889635￿

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Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK

Protein breakdown in the rumen is generally the breakdown process and assesses the regarded as detrimental to the efficiency of relative importance of different species in the ruminant nutrition, certainly for animals on a light of population densities and the properties relatively high plane of nutrition. Peptides and of the mixed rumen population. amino acids arising from proteolysis are potential nutrients for the growth of rumen microorganisms, but they are also liable to be Proteolytic ruminal microorganisms degraded to ammonia and lost from the rumen. Proteolytic activity occurs in all three main The mixed rumen microbial population has a categories of rumen microorganisms. Bacteria proteolytic activity that is only moderate are mainly responsible for dietary protein compared with other proteolytic micro- breakdown, while ciliate protozoa break down organisms and the host’s own gastric and particulate feed protein of appropriate size and pancreatic secretions, but the length of time also bacterial protein. The key features of that feed material is retained in the rumen rumen proteolytic activity are that it varies means that this activity is able to break down a greatly from animal to animal and from feed to substantial proportion of most dietary proteins feed. The predominant mechanism of peptide (0rskov and McDonald, 1979; Broderick et al, degradation is biphasic, via dipeptidyl 1991). Many strains and species of rumen aminopeptidases which cleave dipeptides from ciliate protozoa, bacteria and anaerobic fungi larger peptides followed by dipeptidase. have been found to be proteolytic, and they Dipeptidyl aminopeptidase activity occurs only contain a variety of different types of proteolytic in Prevotella ruminicola among the common enzymes (table I) (Wallace and Cotta, 1988; rumen microbial species. In contrast, Wallace, 1994). dipeptidase, which cleaves the dipeptide The predominant species of proteolytic products from dipeptidyl aminopeptidase, is bacterium found in the rumen of most animals present in many species, including P. is Prevotella (formerly Bacteroides) ruminicola, ruminicola, and is particularly high in rumen which has been identified as proteolytic in protozoa. Deamination of amino acids is many studies (Blackburn and Hobson, 1962; carried out by a combination of numerous low- Fulghum and Moore, 1963; Hazlewood and activity bacteria and protozoa and a much Nugent, 1978; Wallace and Brammall, 1985) smaller number of high-activity species. Most and which can comprise more than 60% of the ammonia production is probably carried out by flora under some circumstances (Van Gylswyk, the low-activity species, which again include P. 1990). Its cell-associated, mainly cysteine ruminicola, but proliferation of the high-activity protease activity, is fairly typical of the rumen species may be a problem on certain diets. bacterial population as a whole. Some animals The microbiology of protein breakdown in the possess Butyrivibrio fibrisolvens as the most rumen is of interest because it deals with one prevalent proteolytic isolate (Blackburn and of the major inefficiencies of ruminant nutrition, Hobson, 1962; Fulghum and Moore, 1963; namely the too-rapid conversion of protein to Hazlewood et al, 1983; Wallace and Brammall ammonia in the rumen and the subsequent 1985). B. fibrisolvens, which has a higher loss of that ammonia by absorption across the specific activity than most other species rumen wall and excretion as urea (Leng and (Wallace and Brammall, 1985), appeared to be Nolan, 1984; Wallace and Cotta, 1988; present when the dietary protein was more Wallace, 1994). This review describes the resistant to degradation (Wallace et ai, 1987). microorganisms involved at different stages of Its extracellular serine protease activity

(Wallace and Brammall, 1985; Cotta and acrylamide gels (Wallace and Cotta, 1988). Hespell, 1986; Strydom et al, 1986) is not Rational manipulation of ruminal proteolysis by typical of in vivo activity. altering the proteolytic population therefore Rumen ciliate protozoa exhibit a variety of appears only a distant possibility. protease activities, the most important of which are cysteine and aspartic proteases (Forsberg et al, 1984) that have a mixture of specificities Peptidolytic rumen microorganisms with a significant trypsin-like activity (Prins et al, 1983; Forsberg et al, 1984). An Isotricha sp. Peptide breakdown to amino acids must occur had a protease profile on gel electrophoresis for the amino acids to be incorporated into that was quite different to the profile of microbial protein, and when there is sufficient Dasytricha ruminantium, and representatives of energy available to fuel biosynthesis, amino four different entodiniomorphid genera also acids will be incorporated and peptide had different protease patterns (Lockwood et breakdown would not be considered to be a al, 1988). Extracellular extracts of rumen major inefficiency in fermentation. However, ciliates were reported to have a higher activity when energy is unavailable, or when the rate of than intracellular extracts (Shinchi et al, 1986), peptide breakdown exceeds the rate at which it but although these enzymes have been can be assimilated, peptide catabolism leads to characterised biochemically (Shinchi and excessive ammonia production and poor N Kandatsu, 1983; Shinchi et al, 1986), their retention. relevance to the mixed ecosystem has not The great majority of peptidase activity in been established. rumen fluid is aminopeptidase (Wallace et al, Protozoa do not hydrolyse soluble protein 1990a). It is characterised by dipeptides rather as readily as do the bacteria. In the studies of than single amino acids being cleaved from the Ushida et al (1986, 1991 ), defaunation resulted peptide chain (table II) (Wallace et al, 1990a, in a higher activity of rumen fluid towards 1993). Enzymes of this nature are classified as soluble protein, which was believed to be dipeptidyl aminopeptidases (Webb, 1992). The caused by increased numbers of more active main mechanism of hydrolysis in intact rumen bacteria. A similar trend was seen with sheep microorganisms appeared, from the hydrolysis which had been ciliate-free from birth (Wallace of diagnostic synthetic substrates, to be et al, 1987). However, protozoa ingest protein dipeptidyl aminopeptidase type I (DAP-1 ) particles, either in the form of feed particles or (Wallace and McKain, 1989), although other bacteria, and they may be of great significance activities were also apparent. This pattern in diets containing particles of the correct differs from that obtained with sonicated dimensions. bacteria, which indicated among others a Reports of proteolytic activity associated strong X-Ala-p-nitroanilide arylamidase (Ala- with rumen anaerobic fungi are conflicting. DAP) activity (table II). The dipeptides released Whereas Neocallimastix frontalis had a high as a result of DAP activity are then broken specific activity metalloprotease (Wallace and down by separate dipeptidase activity. Ruminal Joblin, 1985), most other fungal isolates peptide breakdown is therefore a two-stage had little proteolytic activity, although process. aminopeptidase was present in all species It was established by comparing the (Michel et al, 1993). Experiments with specific activities of mixed rumen protozoa and gnotobiotic lambs indicate that fungi play a mixed bacteria prepared from rumen fluid that minor role in proteolysis in vivo (Bonnemoy et bacteria were mainly responsible for the al, 1993). breakdown of larger peptides (Newbold et al, Proteolytic activity and the microbial 1989; Wallace et al, 1990c). When pure species responsible for that activity are heavily cultures of the most common rumen bacteria diet-dependent (Nugent and Mangan, 1981; were screened for dipeptidyl aminopeptidase Siddons and Paradine, 1981; Hazlewood et al, activity, remarkably the only common bacterial 1983). However, perhaps the most remarkable species that possessed DAP-1 and had high feature of proteolysis is its inherent variability : Ala-DAP activity was P. ruminicola (table 111) different animals on the same or similar diets (Wallace and McKain, 1991). Selective and housed together had completely different isolation, using fluorogenic DAP-1 substrate patterns of proteolytic enzymes in poly- confirmed this unusual finding. P. ruminicola, like the mixed rumen population, had low Russell et al, 1988, 1991) calculated that these aminopeptidase activity against amino acyl-p- bacteria did not have sufficient activity to nitroanilide substrates but high activity against account for observed rates of ammonia dipeptidyl-p-nitroanilide substrates; it also production by the mixed population in their cleaved dipeptides from longer peptides as the cattle, and isolated a group of bacteria that first step in peptide hydrolysis (Wallace et al, were much less numerous than the others, but 1993). Once again, the pattern of peptide which possessed a specific activity of ammonia breakdown would be expected to change with production which was an order of magnitude diet and its influence on numbers of P. greater than that of the other species. ruminicola. If organisms such as S. bovis were Moreover, these bacteria, unlike the others, to prevail, their leucine aminopeptidase activity were highly sensitive to monensin, and since would predominate and amino acids might be ammonia concentrations are lower when cleaved singly from the peptide chain rather in ruminants receive this dietary ionophore, it was pairs (Russell and Robinson, 1984; Wallace deduced that they must be significant ammonia and Brammall, 1985). However, in the sheep producers in vivo. The species isolated, that have been examined at the Rowett Peptostreptococcus anaerobius, Clostridium Research Institute, there is no doubt that, in sticklandii and Clostridium aminophilum total contrast to proteolysis being carried out by (Paster et al, 1993), were atypical of the main a large and variable number of species, ruminal species, although a large number of oligopeptides are cleaved predominantly by clostridia have been isolated from the rumen only one bacterial species, namely P. over the years (Stewart and Bryant, 1988). ruminicola. These bacteria, and also the mimosine In contrast to the limited occurrence of degrader, Synergistes jonesii (Allison et al, dipeptidyl aminopeptidase, many species of 1992; McSweeney et al, 1993), did not ferment protozoa and bacteria have dipeptidase activity sugars but used amino acids as their main (table 111). Among the bacteria, P. ruminicola source of carbon and energy as well as as a had activity against a wide range of dipeptides, nitrogen source. The dichotomy is, therefore, while M. elsdenii also had a high activity that amino acid deamination could be carried (Wallace and McKain, 1991 The P. ruminicola out predominantly numerically abundant dipeptidase is a Mn-metalloprotease (Wallace bacteria each having low activity, or by et al, 1995). relatively few species each with exceptionally high deaminative activity (Figure 1). Until now, there have been no reports of Rumen microorganisms forming high-activity bacteria similar to those isolated at ammonia from amino acids Cornell having been isolated elsewhere. Recently, however, cattle and sheep at the Many experiments were done in the 1950s, Rowett Research Institute were sampled and 60s and 70s to determine the metabolism and the numbers of monensin-sensitive Trypticase fate of amino acids in mixed rumen contents degraders enumerated (Eschenlauer, 1994). (Blackburn, 1965; Allison, 1970; Chalupa, Their viable count indicated that bacteria 1976; Broderick and Balthrop, 1979). As well capable of growth on Trypticase alone were as being nutritionally wasteful, the products of present at 0.7% of the total bacterial amino acid breakdown may be toxic to the population, numbers similar to those reported animal (Carlson et al, 1972; Onodera, 1993). by Yang and Russell (1993), and like the Most recently the main issue has been the Cornell bacteria they were monensin-sensitive. nature of the microbial population that is However, most of these bacteria, unlike primarily responsible for ammonia production the Cornell isolates, fermented sugars. in vivo. For many years, it had been assumed Furthermore, ammonia production in the rumen that deamination was carried out by a large fluid from which they were derived was much number of the main species of rumen bacteria lower than in the Cornell studies and therefore that had been identified to produce ammonia it was not necessary to invoke the intervention from protein or protein hydrolysates (Bladen et of high-activity bacteria. This may be true in al, 1961). other studies too where the rates of ammonia However, Russell and his colleagues at production are relatively low. Cornell (Chen and Russell, 1988, 1989; Ammonia production in the rumen fluid of these same animals was inhibited less than since the presence of only a small population half by monensin, similar to the inhibitions of these organisms could have a major impact observed in similar sheep with monensin and on the efficiency of N retention by the animal. tetronasin (Wallace et al, 1990b). Yang and Russell (1993) also found that the rate of Conclusions ammonia production from casein was inhibited less than half by extremely high concentrations The pattern of conversion of protein to of monensin (5 mM cf. a likely concentration in ammonia is rather like a funnel. Many species vivo of 4 mM) (Wallace et al, 1981 although participate in the initial proteolytic cleavage. the effect was greater for lower rates of The resultant oligopeptides then funnel down ammonia production. Decreased ammonia to to be hydrolysed largely by one species, P. production with ionophores may indeed be due ruminicola. Thereafter, the as to elimination of products diverge partly high-activity bacteria, of bacteria and break but it should also be that the many species protozoa recognised down dipeptides and amino acids. Regulation influence of ionophores extends beyond those of the process has thus far concentrated on the species whose growth is inhibited and are first or last steps. Since the constriction in the generally recognised to be ’monensin- flow is at P. ruminicola, it seems that the sensitive’. The deaminative of P. activity of this deserve detailed ruminicola and Ruminobacter peptidases organism amylophilus, study. bacteria that can grow when ionophores are present, was greatly diminished when they were grown with ionophores in the medium Literature cited (Newbold et al, 1990). Furthermore, the deamination of reduced amino acids will be Allison MJ (1970) Nitrogen metabolism of ruminal In: of and affected monensin, via decreased micro-organisms. Physiology Digestion indirectly by Metabolism in the Ruminant (Phillipson AT, ed) hydrogenase activity (Russell and Martin, Oriel Press Ltd, Newcastle, 456-472 1984; Hino and Russell, The 1985). long-term Allison MJ, Mayberry WR, McSweeney CS, Stahl DA effects of on ammonia ionophores production (1992) Synergistes jonesii, gen. nov., sp. nov., a in vivo are therefore due partly to an effect on rumen bacterium that degrades toxic the residual, apparently ionophore-insensitive pyridinediols. System Appl Microbiol 15, 522-529 species as well as to the suppression of the Blackburn TH (1965) Nitrogen metabolism in the high-activity species. rumen. 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