Formation of Lysine from α,ε-Diaminopimelic Acid and Negligible Synthesis of Lysine from Some Other Precursors by Rumen Ciliate Protozoa

The possibility of lysine formation from α,ε-diaminopimelate (DAP), acetate, aspartate or α-aminoadipate (AAA) in rumen ciliates was examined. DAP-1,7-¹⁴C added to the medium was decarboxylated and converted to radioactive lysine in great amounts and radioactive pipecolate in small amounts by rumen ciliates. Difference of the ability to form lysine from DAP between genus Entodinium and Diplodinium was not observed. With sodium acetate-U-¹⁴C, amino acids fraction of the supernatant fluid of the incubation medium and ciliates contained only 0.56 and 0.59% of the total radioactivity, respectively. In the case of l-aspartate-U-¹⁴C, 95.1% of the radioactivity of the supernatant fluid desalted and 62.2% of the radioactivity incorporated into ciliates (1.5% of the total radioactivity) remained as aspartate. Autoradiograms revealed the negligible spots of lysine in ciliates in both cases. AAA-6-¹⁴C remained almost unchanged, even after incubation with rumen ciliates.     

In Vitro Metabolism of the Stereoisomers of 2,6-Diaminopimelic Acid by Mixed Rumen Protozoa and Bacteria

Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria. Received: 16 March 1996 / Accepted: 14 May 1996     

Interactions between substrate,fermentation end-products,buffering systems and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro

Summary In animal nutrition, incubation of feed samples with CO₂/HCO₃-buffered rumen fluid is used to predict the nutritional values of the feed. During fermentation, volatile fatty acids (VFAs) are produced, which release CO₂ from the buffer through their H⁺ ions. This indirect gas production amounted to 20.8 ml gas per mmol VFA. By incubating glucose, rice starch and cellulose, the relationship between direct and indirect gas production in relation to fermentation kinetics was studied. The total amount of gas formed was found to be dependent on the composition of the fermentation end-products formed. This could be described by: ml gas = M_v·mmol HAc + 2M_v·mmol HB + 0.87M_v·mmol Tot. VFA where HAc = acetic acid; HB = butyric acid; and M_v = molar gas volume. No clear relationship was found between the rate of fermentation and total gas production. From rice starch more total gas was produced than from glucose and cellulose, which were fermented faster and slower, respectively. Correspondence to: S. F. Spoelstra     

Formation of Lysine from, α, ε-Diaminopimelic Acid Contained in Rumen Bacterial Cell Walls by Rumen Ciliate Protozoa

Cell walls containing α,ε-diaminopimelate-l,7-¹⁴C (DAP) was prepared from Escherichia coli isolated from the rumen. After incubation of ciliates with the cell walls, 22.0% of DAP contained in cell walls of E. coli was converted to lysine and pipecolate. Heat-treated mixed rumen bacteria and heat-treated cell walls of mixed rumen bacteria added to the culture medium of rumen ciliates increased 0.572 and 0.934 μmole/ml of sum of lysine and pipecolate, respectively.

From these results, it is clear that rumen ciliate protozoa can form lysine from DAP contained in the mucopeptide of bacterial cell walls. One of the nutritional significance of inhabitation of ciliates in the rumen was revealed.     

Effects of salinomycin and vitamin B(6) on the in vitro synthesis of lysine from the stereoisomers of 2,6-diaminopimelic acid by mixed rumen protozoa,bacteria, and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (pyridoxine hydrochloride) (B(6)) on the production of lysine from the three stereoisomers of 2,6-diaminopimelic acid (DAP-SI) by mixed rumen protozoa (P), mixed rumen bacteria (B), and their mixture (PB). P, B, and PB were isolated from the rumen of goats given a concentrate mixture and lucerne cubes, separately incubated for 12 h with and without DAP-SI (5 mM) as a substrate and SL (5 &mgr;g/ml) and/or B(6) (10 &mgr;g/ml) as additives. In P suspensions, SL and B(6) reduced the amount of DAP-SI by 2.1 times (p<0.001, where p is probability) and 19.9% (p<0.05), respectively, and also increased the production of lysine by 2.4 times (p<0.001) and 26.8% (p <0.05), respectively, during 12 h incubation. In B suspensions, the reductions of DAP-SI with a single addition of SL or B(6) were 8.5% (p<0.001) and 2.7%, respectively, and lysine production increased by 54.3 and 32.9% (p<0.001), respectively, during 12 h incubation. In PB suspensions, the reductions of DAP-SI were 21.9 and 11.7% (p<0.001) with a single addition of SL or B(6), respectively, and the production of lysine increased by 81.4 and 39.4% (p<0.001), respectively, during 12 h incubation. When SL and B(6) were added together to the P, B, and PB suspensions, lysine production further increased by 12.3, 21.3, and 12.4% more than the cases of adding SL only during 12 h incubation, respectively. SL and B(6) were demonstrated to enhance the production of lysine from DAP-SI by mixed rumen protozoa, mixed rumen bacteria and their mixture in this study.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Effect of soluble maillard reaction products on rumen microorganisms

After incubation of Maillard reaction polymers (MRPs) with rumen fluid from wethers neither volatile fatty acids nor lactate were produced. Soluble polymeric products of the Maillard reaction were nonmetabolizable by a mixed culture of rumen microorganisms. MRPs added at 0.5 and 2 g/L inhibited the growth of seven ruminal Gram-negative bacteria by 20 and 30%, respectively. In four strains of Gram-positive bacteria, MRPs lowered the cell concentration by 11% (0.5 g/L) and 25% (2 g/L). The rumen fungusOrpinomyces jojonii also did not metabolize soluble MRPs.     

The hydrogenation of unsaturated fatty acids by five bacterial isolates from the sheep rumen, including a new species.

Five strictly anaerobic bacteria able to hydrogenate unsaturated fatty acids were isolated from sheep rumen. One was characterized as Ruminococcus albus, two as Eubacterium spp. and two as Fusocillus spp., one of which is named as a new species. The Fusocillus organisms were able to hydrogenate oleic acid and linoleic acid to stearic acid, and linolenic acid to cis-octadec-15-enoic acid. The R. albus and the two Eubacteria did not hydrogenate oleic acid but converted linoleic and linolenic acids to a mixture of octadecenoic acids; trans-octadec-II-enoic acid predominated but several isomeric cis and trans octadecenoic acids were produced together with isomers of non-conjugated octadecadienoic acids. The intermediate and final products of hydrogenation by each organism were compatible with the results from mixed rumen bacteria.     

Biochemical changes in infection droplets containing spores of Botrytis spp. Incubated in the seed cavities of pods of bean (Vicia faba L.)

Infection droplets containing spores of Botrytis cinerea become inhibitory to the growth of germ tubes of the fungus within 18 hr. of their incubation in bean pods. The inhibition is caused by an ether-soluble substance, which has been partially purified, and which counteracts the stimulatory effect of sucrose, glucose, fructose, galacturonic acid and several amino acids, which are also present in the infection droplets. Changes in concentration of these substances have been described in the first 24 hr. after placing infection droplets in pods. The only major difference between droplets containing B. fabae and B. cinerea concerns the nature of the ether-soluble substances produced. Following B. fabae infection a biologically inactive u.v. absorbing substance appears in high yield in place of the antifungal substance formed following B. cinerea infections.     

Effect of Chloral Hydrate on Methane and Propionic Acid in the Rumen

Methane production from pyruvate by mixed rumen bacteria in vitro was nearly totally inhibited by chloral hydrate (0.1 mumole/ml of incubation fluid). This effect was accompanied by an accumulation of gaseous hydrogen and an increase in propionic acid production. Infusion of chloral hydrate (4 g/day) into the rumen of a sheep produced the same effects. Evidence is presented for a direct toxic effect of chloral hydrate upon methane bacteria. Results are discussed in terms of fermentation balances.     

In vitro production of lysine from 2,2'-diaminopimelic acid by rumen protozoa.

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu and Masson & Ling show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10(5) protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.     

In Vitro Production of Lysine from 2,2'-Diaminopimelic Acid by Rumen Protozoa

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu [12] and Masson & Ling [9] show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10⁵ protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.     

The liberation of amino acids and reducing compounds by plant roots

Summary Drying and remoistening of different plants grown in sand and in a sand-soil mixture resulted in the liberation of appreciable amounts of amino acids and detectable amounts of reducing compounds. Small quantities of amino acids were produced even under normal conditions of growth. The amino acids produced could be utilized for growth by amino acid requiring bacteria isolated from soil.Peas grown under aseptic conditions also yielded small amounts of ninhydrin-positive substances. Wheat under similar conditions produced less of such materials but in addition liberated detectable amounts of a reducing compound.These results are discussed in relation to the incidence of amino acid requiring bacteria in the rhizosphere.Contribution No. 385     

Formation of biologically active substances by rhizosphere bacteria and their effect on plant growth

Nine out of seventeen strains of bacteria with a pronounced effect on seed germination and on seedling growth, isolated from root surfaces and rhizosphere soil of maize, were selected for a study on the formation of biologically active substances. β-Indole acetic acid (45–72 μg/1.000 ml) was produced by four strains, gibberelline-like substances (1.0–60.0 μg/1.000ml) by all strains, biotin and pantothenic acid by the majority of strains and nicotinic acid by five strains. Amino acids were formed by all strains but in low amounts. Four strains produced growth inhibitors. The highest amounts of biologically active substances were found in cultures ofPseudomonas fluorescens andBacillus brevis. The various cultures ofPseudomonas fluorescens differed in their capability to produce biologically active substances. The majority of bacterial cultures or their supernatants significantly stimulated the germination of seeds and some of them significantly affected the growth of plants. Inoculation of maize seeds with strainsPseudomonas fluorescens andChromobacterium violaceum significantly increased the yield of dry matter of plants.     

Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.     

Perturbation dynamics of the rumen microbiota in response to exogenous butyrate

The capacity of the rumen microbiota to produce volatile fatty acids (VFAs) has important implications in animal well-being and production. We investigated temporal changes of the rumen microbiota in response to butyrate infusion using pyrosequencing of the 16S rRNA gene. Twenty one phyla were identified in the rumen microbiota of dairy cows. The rumen microbiota harbored 54.5±6.1 genera (mean ± SD) and 127.3±4.4 operational taxonomic units (OTUs), respectively. However, the core microbiome comprised of 26 genera and 82 OTUs. Butyrate infusion altered molar percentages of 3 major VFAs. Butyrate perturbation had a profound impact on the rumen microbial composition. A 72 h-infusion led to a significant change in the numbers of sequence reads derived from 4 phyla, including 2 most abundant phyla, Bacteroidetes and Firmicutes. As many as 19 genera and 43 OTUs were significantly impacted by butyrate infusion. Elevated butyrate levels in the rumen seemingly had a stimulating effect on butyrate-producing bacteria populations. The resilience of the rumen microbial ecosystem was evident as the abundance of the microorganisms returned to their pre-disturbed status after infusion withdrawal. Our findings provide insight into perturbation dynamics of the rumen microbial ecosystem and should guide efforts in formulating optimal uses of probiotic bacteria treating human diseases.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

The uptake of bacteria and amino acids by Ophryoscolex caudatus, Diploplastron affine and some other rumen Entodiniomorphid protozoa

The rate of uptake of mixed rumen bacteria and free amino acids by washed suspensions of seven species of rumen ciliate protozoa has been followed. By assuming that the behaviour of these protozoa was the same under these conditions as during growth it was shown that Ophryoscolex caudatus could obtain the amino acids for growth by the engulfment of rumen bacteria. However, all the cellulolytic protozoa studied (Diploplastron affine, Diplodinium anacanthum, Diplodinium anisacanthum, Enoploplastron triloricatum, Eremoplastron bovis and Ostracodinium obtusum bilobum) were unable to obtain sufficient amino acids from either source to grow at even 25% of the maximum rate and it is postulated that they might utilize plant protein. O. caudatus grown in vitro did not engulf Klebsiella aerogenes or Escherichia coli but took up other bacteria and a rumen yeast at rates of up to 54000 organisms/protozoon/h from a population density of 10⁹/ml. When grown in vivo it was more selective and engulfed mixed rumen bacteria at only 10% of the rate obtained with protozoa grown in vitro. D. affine grown in vitro did not engulf Bacteroides ruminicola, Esch. coli, Kl. aerogenes or Proteus mirabilis but took up mixed rumen bacteria from a population of 10⁹/ml at a rate of 2200 bacteria/ protozoon/h.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Effect of adsorbants on in vitro biohydrogenation of 22:6n-3 by mixed cultures of rumen microorganisms

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.