CURRENT MICROBIOLOGY Vol. 26 (1993), pp. 333-336 Current Microbiology �9 Springer-Verlag New York Inc. 1993

Breakdown of Different Peptides by Prevotella (Bacteroides) ruminicola and Mixed Microorganisms from the Sheep Rumen

R. John Wallace, 1 Nest McKain] and Glen A. Broderick 2

IRowett Research Institute, Bucksburn, Aberdeen, UK; and 2U.S. Dairy Forage Research Center, Madison, Wisconsin, USA

Abstract. Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells of Prevotella (formerly Bacteroides) ruminicola M384 and P. ruminicola Bl4. The rates of break- down of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline- containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides by P. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.

Rumen microorganisms convert much of the protein nitrogen entering the rumen to ammonia [6, ! 1]. The rate of ammonia production usually exceeds the ca- pacity of the microbial population to assimilate am- monia nitrogen, and the excess ammonia tends to diffuse across the rumen wall, finally being excreted as urea. Factors governing the rate at which ammo- nia is produced are, therefore, relevant to our under- standing of this nutritionally wasteful process.

Peptides are intermediates in the conversion of protein nitrogen to ammonia. The structure of the N-terminus of individual peptides had a major influ- ence on the rate at which the peptide was hydrolyzed in rumen fluid, with proline causing the peptide to be degraded more slowly [1, 18]. An isopropyl alcohol extraction of Trypticase yielded hydrophobic pep- tides that produced ammonia more slowly than the residual hydrophilic peptides [3], but no evidence of a similar relation between the rate of peptide breakdown and their hydrophobicity was observed with individual pure peptides [1, 18].

Ciliate protozoa have a higher dipeptidase activ- ity than rumen bacteria [ 17], although many bacterial species hydrolyze Ala2 [15], and bacterial dipepti- dase activity replaces protozoal activity when the protozoa are absent [16]. The main hydrolytic activ- ity towards higher peptides in vivo is bacterial and cell associated [17]. The breakdown pattern of ala-

nine peptides [1, 14, 17, 18] and arylamide substrates [14] suggested that the main bacterial activity was a dipeptidyl aminopeptidase type 1 (DAP-1) mecha- nism. A survey of laboratory cultures [15] and selec- tive isolations from rumen fluid [9] both indicated that the only predominant species to possess DAP- 1 was Prevotella (formerly Bacteroides) ruminicola, even in strains with widely divergent Chromosomal homologies [7].

The aims of this work were to determine whether other peptides were broken down by a simi- lar dipeptidyl aminopeptidase mechanism, whether two distantly related strains of P. ruminicola had peptidolytic properties similar to those of mixed ru- men microorganisms, and what structural features of peptides determine their rate of breakdown in rumen fluid and by P. ruminicola.

Materials and Methods

Incubations with rumen fluid. Two mature wether sheep, fitted with permanent rumen fistulae, were fed 450 g of a mixed diet twice daily at 0800 and 1600 h. The diet consisted of 67% grass hay and 33% concentrate [19]. Strained rumen fluid was prepared 2-3 h after the morning feeding by straining rumen contents through two layers of muslin.

Strained rumen fluid (0.8 ml) was added to 0.2 ml of 1.25 mM peptide solution and the mixture was incubated at 39~ The reaction was stopped by adding 0.25 ml of 1.25 M orthophosphoric acid and the mixture was centrifuged at 12,000 g for 5 rain. The

Address reprint requests to: Dr. R.J. Wallace, Rowett Research Institute, Bucksburn, Aberdeen. AB2 9SB, UK.

334 CURRENT MICROBIOLOGY Vol. 26 (1993)

Table 1. Breakdown of dipeptides by Prevotella ruminicola strains M384 and Bl4 and mixed rumen microorganisms

Rate of breakdown (nmol/min per mg protein)

P, ruminicola P. ruminicola Peptide Sheep 1 Sheep 2 M384 B14

GlyPro 0.16 0.21 0.70 0.32 AspLys 0.20 0.39 - - - - AspGlu 0.29 0.24 - - - - GlyAla 0.40 0.63 - - - - AspGly 0.44 0.43 - - - - LysAsp 0.44 0.87 1.11 0 ValTyr 0.58 0.28 6.41 5.06 GlyHis 0.58 0.66 4.51 3.22 SerGlu 0.60 1.18 0.95 1.92 GlyAsp 0.65 0.43 - - - - ValGly 0.65 0.60 6.41 0.92 GluLys 0.73 0.63 - - - - ValAla 0.74 0.71 6.04 3.01 LeuGly 0.74 1.01 - - - - GlyPhe 0.80 0.58 2.90 6.01 HisLys 0.88 1.07 5.81 6.86 GlyGly 0.97 0.74 4.99 1.29 HisGly 0.97 0.89 - - - - AlaAla 1.11 0.70 4.62 25.63 GlySer 1.53 1.87 4.56 2.16 AlaPhe 1.62 0.89 10.70 13.61 AlaGly 1.69 0.94 4.60 3.92

supernatant fluid was analyzed for peptides by HPLC. All incuba- tions were carried out in duplicate.

Incubations of peptides with P. ruminicola. Fresh overnight cul- tures ofP. ruminicola strains M384 [13] and BI4 [2] were grown in Hungate tubes on rumen fluid-containing general purpose medium [5]. Cells were harvested by centrifuging the tubes at 1100 g for 10 rain, the supernatants were discarded, and the pellets were washed once in anaerobic 25 mM potassium phosphate buffer, pH 7.0, and resuspended in half (M384) or the original (B14) volume of the same buffer. Under COz, 0.8 ml of suspension was added to 0.2 ml of 1.25 mM peptide, and the mixture was incubated under CO 2 at 39~ The reaction was stopped and the mixture centrifuged as for rumen fluid incubations. The results are means of duplicate incubations.

Analyses. Peptides were analyzed by ion-pairing reverse-phase HPLC as described previously [14]. Protein was measured with the alkaline extraction and Folin reagent procedure described by Herbert et al. [4].

Results and Discussion

D i p e p t i d e s i n c u b a t e d wi th r u m e n fluid in v i t ro we re h y d r o l y z e d at r a t e s wh ich r anged f rom 0.16 to 1.87 nmol /min p e r mg p r o t e i n (Table 1). The r ank ing or- d e r for d i f fe ren t p e p t i d e s was s imi la r in the two sheep s a m p l e d . G l y P r o was m o s t s lowly hy-

Table 2. Breakdown of tripeptides by P. ruminicola stratus M384 and B14 and mixed rumen microorganisms

Rate of breakdown (nmol/min per mg protein)

P. ruminicola P. ruminicola Peptide Sheep 1 Sheep 2 M384 B14

AlaAlaAla 3.30 3.41 5.04 23.26 GlyHisLys 0.81 0.88 6.25 3.53 LeuGlyGly 0.56 0.82 2.15 1.76 GlyGlyPhe 0.43 0.52 0.92 0.95

d r o l y z e d , c o n s i s t e n t wi th the w i d e r r e s i s t a n c e to d e g r a d a t i o n o f P r o - c o n t a i n i n g p e p t i d e s [1, 18]. Di- p e p t i d e s con ta in ing A s p were a lso m o r e r e s i s t a n t to b r e a k d o w n , again in a c c o r d wi th p r e v i o u s obse r - va t ions tha t ac id ic p e p t i d e s , inc lud ing d i p e p t i d e s and o l i gopep t ide s , t e n d e d to be m o r e s l owly b r o k e n d o w n than neu t ra l or bas i c p e p t i d e s [18].

G l y P r o was a lso s l owly d e g r a d e d b y b o t h s t ra ins o f P. ruminico la , bu t o t h e r w i s e the r ank ing o r d e r was s o m e w h a t d i f fe ren t f rom m i x e d r u m e n fluid (Table 1). The d i p e p t i d a s e a c t i v i t y o f P . ruminico la was h igher than tha t o f r u m e n fluid in all c a se s e x c e p t L y s A s p wi th s t ra in B~4, and wi th mos t p e p t i d e s the ra te o f h y d r o l y s i s was s e v e r a l t imes h igher in P. rumin ico la . The two s t ra ins we re m o r e s imi lar to each o t h e r t han to r u m e n fluid, wi th a few e x c e p t i o n s . A l t h o u g h P. rumini-

cola, t o g e t h e r wi th s e ve ra l o t h e r spe c i e s o f t u r e e n b a c t e r i a [15], h y d r o l y z e s d i p e p t i d e s , it is a c tua l l y the c i l ia te p r o t o z o a w h i c h a re m o s t ac t ive aga ins t d ipe p t i de s in the m i x e d p o p u l a t i o n [17]. Thus , the rank ing o r d e r o f d i f fe ren t d i p e p t i d e s wi th r u m e n fluid p r o b a b l y ref lec ts the i r s u s c e p t i b i l i t y to p r o t o - zoa l r a the r than b a c t e r i a l a t t ack .

The ra te o f h y d r o l y s i s o f t r i p e p t i d e s was s imi la r in s a mp le s o f r u m e n fluid t a k e n f rom b o t h s h e e p , bu t again the p a t t e r n was d i f fe ren t in the two s t ra ins of P. rumin ico la (Tab le 2). The r ank ing o r d e r o f t r i pep t ide h y d r o l y s i s b y P. rumin ico la B~4 was the s ame as tha t o f r u m e n fluid. B o t h s t ra ins had h igher t r i p e p t i d a s e a c t i v i t y than tha t o f r u m e n fluid. T o o few t r i p e p t i d e s w e r e e x a m i n e d to m a k e fu r the r c o n c l u s i o n s a b o u t s t r u c t u r e / d e g r a - dab i l i ty r e l a t ions , a l t hough the G l y G l y g r o u p at the N - t e r m i n u s was m o s t s l owly d e g r a d e d in r u m e n fluid and b y P. ruminico la , as was o b s e r v e d pre - v ious ly in i n c u b a t i o n s wi th r u m e n fluid in v i t ro

[18]. T e t r a p e p t i d e s i n c u b a t e d wi th r u m e n fluid w e r e

b r o k e n d o w n in a m a n n e r c o n s i s t e n t wi th c l e a v a g e in

R.J. Wallace et al.: Peptide Breakdown by Rumen Microorganisms 335

Table 3. Breakdown of tetrapeptides by mixed rumen microorganisms

Peptide concentration (nmol/ml) Time (min) Sheep 1 Sheep 2

Ala4 Ala3 Ala2 Ala4 Ala3 0 250 0 0 250 0 5 36 0 373 126 2

10 0 2 373 101 3 30 0 0 105 10 0 60 3 0 10 13 0

ValAla2Phe ValAla Ala 2 AlaPhe ValAla2Phe ValAla 0 250 0 0 0 250 0 5 64 134 0 160 131 71

10 0 117 0 150 67 138 30 0 73 0 50 0 184 60 0 28 0 10 0 132

ValGlySerGlu ValGly GlySer SerGlu ValGlySerGlu VatGly 0 250 6 0 0 250 0 5 190 56 0 18 165 1

10 136 136 0 38 123 28 30 26 129 0 31 43 173 60 2 42 0 0 7 79

Ala 2 0

206 310 251 163

Ala 2 0 0 0 0 0

GlySer 0 0 0 0 0

AlaPhe 0

102 161 143 82

SerGlu 0

33 40 72

0

Table 4. Breakdown of larger peptides by P. ruminicola M384 and B~4

Peptide concentration (nmol/ml) Time (min) P. ruminicola M384 P. ruminicola B14

Ala4 Ala3 Ala2 Ala4 Ala3 Ala2 0 250 0 0 250 0 0

10 206 0 36 0 32 210 20 185 0 53 0 18 67 30 155 0 67 0 13 18 60 85 0 88 0 0 0

ValAla~Phe ValAla Ala 2 AlaPhe ValAla2Phe ValAla Ala2 0 250 0 0 0 250 0 0

10 209 18 0 12 5 205 0 20 169 28 0 17 0 213 0 30 135 38 0 18 0 164 0 60 65 42 0 14 0 131 0

VNGIySerGlu ValGly GlySer SerGlu ValGlySerGlu ValGly GlySer 0 250 0 0 0 250 0 0

10 232 0 0 8 56 81 0 20 224 3 0 15 21 128 0 30 213 10 0 24 7 208 0 60 192 21 0 41 0 243 0

ArgLysAspValTyr ArgLys LysAsp AspVal ValTyr ArgLysAspValTyr ArgLys LysAsp 0 250 0 0 0 0 250 0 0

10 217 23 0 0 15 12 48 0 20 196 38 0 0 21 0 82 0 30 163 46 0 0 25 0 101 0 60 112 73 0 0 27 0 128 0

AlaPhe 0

250 216 148 53

SerGlu 0

142 206 250 250

AspVal ValTyr 0 0 0 130 2 169 3 140

30 128

336 CURRENT MICROBIOLOGY Vol. 26 (1993)

the middle of the molecule, releasing two dipeptides (Table 3). Whether the cleavage occurred from the N- or C-terminus is not clear from this experiment, but previous experiments showed that blocking the N- terminus of ValGlySerGlu with an acetyl group inhib- ited its degradation completely in a 6-h incubation [12]. This pattern is consistent with the conclusion based on the breakdown pattern of alanine peptides, ArgLysAspValTyr , and arylamides [14, 17, 18] that the main mechanism of hydrolysis in the rumen is a dipeptidyl aminopeptidase [8]. Both strains of P. rum- inicola also produced dipeptides from the N-terminus of higher peptides (Table 4), suggesting a similar mechanism for P. ruminicola. The results of earlier experiments with alanine peptides and synthetic sub- strates had pointed to a similar conclusion [ 15]. Man- narelli et al. showed that chromosomal D N A from strains M384 and BI4 gave less than 20% hybridiza- tion, indicating that they were only distantly related. It is remarkable, therefore, that they are so similar in their peptide metabolism.

Several observations imply that, although di- peptidyl aminopeptidase is the predominant mecha- nism for peptide breakdown in the rumen and by P. ruminicola, the reaction may not be simple, and other peptidases are undoubtedly involved. ValGly was released from ValGlySerGlu more slowly than SerGlu with P. ruminicola B14 (Table 4), implying that another intermediate had been formed. This discrepancy was not observed in other incubations. Furthermore, the C-terminal dipeptide ValTyr was formed as well as ArgLys from ArgLysAspValTyr by P. ruminicola M384, and indeed ValTyr was formed more rapidly than ArgLys by strain B14 (Ta- ble 4). N-terminal acetylation of mixed peptides in- hibited their breakdown in vitro only by a maximum of 70%, with several individual peptides being 100% protected but others much less effectively protected [12]. Acetylation did not protect ArgLysAspValTyr from degradation [12]. Thus, it seems likely that ArgLysAspValTyr , and probably other peptides, are broken down by other types of protease or pepti- dase both in the mixed population and in P. rumini- cola. These peptidases must be different from the leucine aminopeptidase activity, which is present at about 30% of dipeptidyl aminopeptidase activity in rumen fluid [14] and which has also been detected in Streptococcus boris [I0, 13, 15].

ACKNOWLEDGMENTS

This work was supported by Royal Gist-brocades nv, Delft, The Netherlands, and the Scottish Office Agriculture and Fisheries Department. We thank Nicola D. Walker for technical assistance.

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