Isolation of proteolytic rumen bacteria by use of selective medium containing leaf fraction 1 protein (ribulosebisphosphate carboxylase).

The principle proteolytic bacteria isolated from bovine rumen contents by virtue of the ability to obtain nitrogen from the proteolysis of leaf fraction 1 protein (ribulosebisphosphate carboxylase, EC 4.1.1.39) were identified as Streptococcus bovis and Butyrivibrio spp. Substitution of fresh fodder, rich in soluble protein, for a hay-concentrates diet resulted in enhanced ruminal proteolytic activity and a significant increase in the number of bacteria able to use fraction 1 protein as the sole nitrogen source. Isolated proteolytic bacteria degraded fraction 1 protein and casein readily. Bovine serum albumin was attacked by Butyrivibrio spp. but was resistant to proteolysis by the streptococci.     

Synergism between different species of proteolytic rumen bacteria

Proteolytic strains ofButyrivibrio alactacidigens, Butyrivibrio fibrisolvens, Selenomonas ruminantium, andStreptococcus bovis grew markedly better in medium containing casein as sole nitrogen source when they were inoculated in pairs rather than singly, when growth was poor or nil. The improved growth ofSelenomonas ruminantium with other species was a consequence of a faster rate of proteolysis by the mixed culture. The cooperativity withStreptococcus bovis was mediated by the extracellular activity ofSelenomonas ruminantium. Synergistic growth betweenBu. alactacidigens and other bacteria was not due to cooperativity between proteolytic activities but probably to nutritional interdependence between the species. Cooperative proteolysis also occurred in mixtures ofSelenomonas ruminantium andBacteroides ruminicola, a proteolytic isolate capable of growth on casein in monoculture.     

Protein degradation by ruminal microorganisms from sheep fed dietary supplements of urea, casein, or albumin

Ruminal fluid from sheep fed hay plus concentrate diets containing 1.8% urea, 6% casein, or 6% egg albumin had proteolytic activities of 4.12, 3.02, or 4.00 mg of [14C]casein hydrolyzed ml-1 h-1, respectively. Dietary albumin had no effect on the rate of albumin breakdown relative to that of casein (0.06). Greater numbers of highly proteolytic bacteria, mainly Butyrivibrio spp., were isolated from the rumens of sheep receiving albumin. Albumin hydrolysis by these isolates was even slower relative to that of casein (0.03) than in ruminal fluid and was similar to that found in isolates from urea- and casein-fed sheep. Hence, there appears to be no mechanism by which ruminal bacteria can alter their proteolytic activity to utilize resistant soluble protein more effectively.     

Protein degradation by ruminal microorganisms from sheep fed dietary supplements of urea, casein, or albumin.

Ruminal fluid from sheep fed hay plus concentrate diets containing 1.8% urea, 6% casein, or 6% egg albumin had proteolytic activities of 4.12, 3.02, or 4.00 mg of [14C]casein hydrolyzed ml-1 h-1, respectively. Dietary albumin had no effect on the rate of albumin breakdown relative to that of casein (0.06). Greater numbers of highly proteolytic bacteria, mainly Butyrivibrio spp., were isolated from the rumens of sheep receiving albumin. Albumin hydrolysis by these isolates was even slower relative to that of casein (0.03) than in ruminal fluid and was similar to that found in isolates from urea- and casein-fed sheep. Hence, there appears to be no mechanism by which ruminal bacteria can alter their proteolytic activity to utilize resistant soluble protein more effectively.     

Protein degradation by human intestinal bacteria

Analysis of human gut contents showed that substantial quantities of soluble protein, ammonia and branched chain volatile fatty acids occurred throughout the large intestine [0.1-24.4 g (kg contents)-1, 7.7-66.0 mmol (kg contents)-1 and 1.5-11.1 mmol (kg contents)-1 respectively]. The presence of these metabolites suggested that substantial proteolysis was occurring. In vitro studies showed that casein and bovine serum albumin were partly degraded in slurries of human faeces over a 96 h incubation period, to produce TCA-soluble peptides, ammonia and volatile fatty acids. Proteolytic activity detected in the stools of five individuals ranged from 3.5 to 19.8 mg azocasein hydrolysed h-1 (g faecal material)-1. Washed cell and washed particulate faecal fractions accounted for 24-67% of total activity. The predominant proteolytic bacteria in the faecal samples examined were identified as Bacteroides spp. [1.0 X 10(11)-1.3 X 10(12) (g dry wt faeces)-1] and Propionibacterium spp. [1.2 X 10(8)-1.0 X 10(10) (g dry wt faeces)-1]. Other proteolytic bacteria which occurred in lesser numbers were identified as belonging to the genera Streptococcus, Clostridium, Bacillus and Staphylococcus. These results demonstrate that the gut microflora could potentially play a major role in proteolysis in the human colon.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p -nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis -type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p -nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N -succinyl alanine alanine phenylalanine proline p -nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio -like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella -like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N -succinyl phenylalanine p -nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.     

Butyrivibrio spp. and other xylanolytic microorganisms from the rumen have cinnamoyl esterase activity

High concentrations of hydroxycinnamic acids in the hemicellulosic fraction of dry season tropical grasses may influence the rate of microbial degradation of arabinoxylans by ruminant animals. The ability of 22 strains of Butyrivibrio fibrisolvens, other ruminal bacteria (Ruminococcus albus SY3, Ruminococcus flavefaciens RF1,Prevotella ruminicola AR20) and the ruminal phycomycete Neocallimastix patriciarum CX to digest the tropical grass Heteropogon contortus(spear grass) and hydrolyse esterified ferulic and p-coumaric acid was examined. Significant digestion (8-36%) of spear grass occurred with the B. fibrisolvens strains H17c, A38, LP92-1-1, 49,R. albus SY3 and N. patriciarum. Hydrolysis of ester-linked ferulic and p-coumaric acid occurred with all organisms except B. fibrisolvens strains GS113, OB156 and LP1028 and P. ruminicola AR20. The ratio of ferulic to p-coumaric acid hydrolysed by different strains of Butyrivibrio spp. varied markedly from 0.96 for AR 51 to 0.16 for A38. Butyrivibrios which were fibrolytic (H17c and A38) had higher extracellular cinnamoyl esterase activity than bacteria that did not digest spear grass fibre (LP 91-4-1 and AR 20) which had low activities or only produced cell associated enzyme. Cell associated and extracellular esterase activity were induced when Butyrivibrio spp. strains H17c, A38 and E14 and the Ruminococcus spp. were grown on birchwood xylan but induction did not occur to the same extent with N. patriciarum. This is the first reported observation of cinnamoyl esterase activity in the genus Ruminococcus. The fungus N. patriciarum had significantly higher digestibility of spear grass and solubilisation of phenolic acids than the bacteria. The study shows that high levels of extracellular cinnamoyl esterases are characteristic of a selection of fibre-degrading ruminal bacteria and fungi which probably indicates that these enzymes are common amongst xylanolytic ruminal microorganisms.     

In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process

Misfolded proteins undergo a preferent degradation ruled by the housekeeping bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases. The susceptibility of aggregated polypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells. We have studied here the in vitro proteolysis of beta-galactosidase fusion proteins by trypsin treatment of purified inclusion bodies. A cascade digestion process similar to that occurring in vivo has been observed in the insoluble fraction of the digestion reaction. This suggests that major protease target sites are not either lost or newly generated by protein precipitation and that the digestion occurs in situ probably on solvent-exposed surfaces of inclusion bodies. In addition, the sequence of the proteolytic attack is influenced by protein determinants other than amino acid sequence, the early digestion steps having a dramatic influence on the further cleavage susceptibility of the intermediate degradation fragments. These observations indicate unexpected conformational changes of inclusion body proteins during their site-limited digestion, that could promote protein release from aggregates, thus partially accounting for the plasticity of in vivo protein precipitation and solubilization in bacteria.     

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Abstract: The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source. Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci. Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane. Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo. This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria.     

Effects of Ammonia and Amino Acids on the Growth and Proteolytic Activity of Three Species of Rumen Bacteria: Prevotella albensis, Butyrivibrio fibrisolvens, and Streptococcus bovis

The addition of increasing physiological concentrations of ammonia or amino acids had distinct effects on the growth and proteolytic activity of Streptococcus bovis JB1, Prevotella albensis, and Butyrivibrio fibrisolvens DSM3071. The growth of S. bovis and B. fibrisolvens was enhanced by NH₃ and AA, and that of P. albensis was reduced compared with a control with protein as the sole source of nitrogen. The proteolytic activity of S. bovis and P. albensis was reduced, but that of B. fibrisolvens was improved. NH₃ seemed to act mainly on the cell-associated fraction of the proteolytic activity, while the action of AA was not specific. In the rumen the proteolytic activity of S. bovis and P. albensis would be optimal at low concentrations of NH₃ or AA (<0.05 and <0.27 g/L respectively). In contrast, B. fibrisolvens would need higher concentrations (0.5 g/L of NH₃ or 2.7 g/L of AA). It can be assumed that these bacteria will grow in different ecological niches. Received: 5 October 1999 / Accepted: 30 December 1999     

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens.

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of a cAMP-binding protein is inhibited by human c-Ha-ras gene products

Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha-ras gene products produced by Escherichia coli. The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha-ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.     

Evidence pointing to the main role of lysosomes in mitochondrial proteolysis at neutral pH

Recombination experiments using radioactive mitochondria and mitoplasts, and nonradioactive lysosomes or digitonin-soluble fraction of mitochondria, show equal rates of proteolysis and of inactivation of carbamyl phosphate synthetase; the amount of lysosomal protein was equal in both cases on the basis of N-acetyl-beta-glucosaminidase activity. Therefore, lysosomes seem to be responsible for all the proteolytic activity exhibited by the digitonin soluble fraction of mitochondrial preparations. Since this fraction contains ca. 90% of the proteolytic activity present in mitochondrial preparations, most of the proteolysis can be attributed to lysosomal contamination. These findings and stability characteristics "in vitro" and "in vivo" of some matrix enzymes are presented and discussed in relation to protein turnover.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors.

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

Effects of exogenous ammonia or free amino acids on proteolytic activity and protein breakdown products in Streptococcus bovis,Prevotella albensis,and Butyrivibrio fibrisolvens

We studied in details the ammonia or free amino acids (AA) effects on proteolytic activity of three ruminal bacteria: enzymatic activities and protein breakdown products were measured at the end of the exponential growth phase. In Streptococcus bovis the simultaneous uptake of ammonia and probably small peptides induced a decline in total proteolytic activity as a result of changes in endopeptidasic activities. With free AA, the tendency for the endopeptidasic activities to specialise was more evident and the total proteolytic activity decreased too. In Prevotella albensis, the inhibition of proteolysis with free AA was linked to the disappearance of free endopeptidases, to the specialization of cell-associated endopeptidases and to the decrease in exopeptidases. The decrease of proteolysis in P. albensis when ammonia was added was more difficult to interpret. With ammonia or AA Butyrivibro fibrisolvens developed a distinct behavior of those expressed by the other species: the increase of the total proteolytic activity could be explained by a better balance of the endopeptidases expressed. It then clearly appeared that the expression of the proteolytic activities are linked to the nature and/or to the quantity of the nitrogen source. This leads each species to adopt its own nutritional strategy in order to adapt to the environmental conditions of the ruminal ecosystem. Received: 2 July 2001 / Accepted: 28 September 2001     

Isolation of intact FNR protein (Mr 30 000) of Escherichia coli

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000. The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29).