Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen

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1 JOURNAL OF BACTERIOLOGY, Jan. 1974, p Copyright 1974 American Society for Microbiology Vol. 117, No. 1 Printed in U.S.A. Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen MILTON J. ALLISON, I. M. ROBINSON, AND A. L. BAETZ National Animal Disease Laboratory, Agricultural Research Service, Ames, Iowa 51 Received for publication 9 February 1973 Microbes in ruminal contents incorporated "C into cells when they were incubated in vitro in the presence of ["C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular "C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate "C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved. Indole-3-acetic acid (IAA) is a major product of tryptophan metabolism by the microbial population of the rumen (16, 3) and is also formed by bacteria from human feces (33). The experiments described in this paper were conducted to test the hypothesis that IAA might be carboxylated by microorganisms in the rumen to synthesize the carbon skeleton of tryptophan. This hypothesis was based on evidence that certain ruminal bacteria synthesize the carbon skeletons of several amino acids by carboxylating acids with one less carbon than the amino acid product. Thus, isovalerate, isobutyrate, 2-methyl-n-butyrate, and phenylacetate are carboxylated in biosynthetic reactions leading to leucine, valine, isoleucine, and phenylalanine, respectively, (1) and succinate is carboxylated to form the carbon skeleton of glutamate (5). A note concerning some of the experiments described here has been published (4). MATERIALS AND METHODS Culture methods. Ruminal ingesta was obtained through ruminal cannulas in adult sheep and a cow fed a ration of alfalfa hay plus grain. Whole ruminal contents, in some experiments, and in others the filtrate that passed through two layers of cheesecloth were incubated in vitro at 39 C under CO2 with added [1-"C]IAA. The rate of incorporation of "C into cellular material was measured by determination of the "C retained on membrane filters (.45 um, type HA; Millipore Corp.). Portions of the fermentation mixture were removed while gasing with CO,, diluted with anaerobic dilution solution (7), and filtered and washed with this solution. In other experiments with whole ruminal contents, a differential centrifugation 175 procedure was used that precipitated most of the protozoa, some bacteria, and some plant material at 3 x g for 5 min. The supernatant fraction was then centrifuged at 25, x g to recover the rest of the bacterial cells. Pure cultures of ruminal bacteria were from the stock collection of M. P. Bryant. With the exceptions listed below, organisms were grown in chemically defined media (8, 28) that did not contain amino acids other than cysteine and methionine. An amino acid mixture that did not contain tryptophan (2) was added to the medium used to culture Peptostreptococcus elsdenii and Butyrivibrio fibrisolvens. Ruminococcus bromii ATCC (24) did not grow in the defined media so experiments with it were conducted using RGCA broth (7) which contained 4% rumen fluid. Fractionation procedures. The methods used to fractionate cells were those of Roberts et al. (27). The hot trichloroacetic acid precipitate or "protein" fractions were usually hydrolyzed (in sealed tubes under N, at 15 C for 2 h) using alkali, usually 14% (wt/ vol) Ba(OH),-8 H,O. After hydrolysis, barium was removed as the carbonate. When NaOH was used for hydrolysis, the neutralized hydrolysate was treated with Dowex 5 H+ (27) prior to chromatography. In some of the later experiments, protein was hydrolyzed with p-toluenesulfonic acid (19). Paper chromatographic systems used to separate amino acids included a butanol-acetic acid system (32) (benzyl alcohol-butanol buffered at ph 6.2 and collidine buffered at ph 9 [22]) and a two-dimensional system (23). Solvents used for thin-layer chromatographic separations of amino acids (26) included propanol-water (7:3) and methyl ethyl ketone-pyridine-water-acetic acid (7:15:15:2) with precoated Sil Plate 22 (Brinkman), and methanol-water-pyridine (2:5: 1) with precoated cellulose plates (E. Merck A.G.). Amino acids from some of the protein

2 176 ALLISON, ROBINSON, AND BAETZ J. BACTERIOL. hydrolysates were also separated by using an amino acid analyzer (Beckman 121). It was necessary to use more than one chromatographic system to have confidence that the labeled product was tryptophan because IAA migrated with or near tryptophan on the ion exchange resin columns used with the amino acid analyzer and on several thin-layer chromatography systems other than those listed above. Labeled compounds. The [1-"CJIAA used was from several different batches (Nuclear Research Chemicals, Inc., and Mallinkrodt Nuclear Corp.). The specific activity of' IAA used in separate experiments is given below. Purity ot' the labeled IAA was checked by preparing radioautographs after thinlayer chromatography on pre-coated Sil N-HR plates (Brinkman) with isopropanol-25'. NH4OH-water (85:5: 15) as developing solvent. Traces of' radioactive impurities that accumulated upon storage were removed from samples of' IAA by using this same system. The DL-[3-`Cjserine (Nuclear Research Chemicals, Inc.) used had a specific activity of 5.23 Ci/mol, and DL-["CJshikimic acid (1-6 ring) (Calbiochem) had a specitic activity of 8.7 Ci/mol. Labeled IAA was dissolved in acetonitrile. This solution was transf'erred to sterile, dry culture vessels, and the acetonitrile was evaporated under a stream of' CO2 before sterile culture media was placed in the vessel. Solutions of' other labeled compounds were filter sterilized. Measurement of radioactivity. Radioactivity was usually measured in a liquid scintillation counting system (Packard Instrument Co.) with the XDC solvent of Bruno and Christian (6). Sodium hydroxide was added to culture supernatant fluids to prevent loss of' "CO2 which was subsequently measured after acidif'ication and diff'usion into p-(diisobutyl-cresoxyethoxyethyl) dimethylbenzyl ammonium hydroxide (21). Specif'ic activity measurements of' carbonate were made after diff'usion into NaOH. Carbonate was precipitated as the barium salt. It was washed, dried, and weighed, and then "C was measured after diff'usion into an ethanolamine trapping solution (12). Counting efficiency was monitored by using external standardization and determined by addition of' ["C ]toluene as an internal standard. Radioactivity on membrane f'ilters (Millipore Corp.) was determined by using a windowless gas flow counting system. The membrane filters were cemented to planchets and dried under an infrared lamp before they were counted. Radioactive areas on paper chromatograms were located by using a chromatogram strip scanning system and preparation of radioautographs. Radioactivity in amino acids that were separated by the amino acid analyzer was measured by passing the eluate from the column through an anthracene packed flow cell (Packard Instrument Co.) prior to reaction with ninhydrin. The signal from the scintillation spectrometer was fed to an analytical count ratemeter and recorded on one channel of a strip chart recorder. Counts from the spectrometer were also fed to an electronic counting circuit which included a digital to analog converter. This gave an output proportional to accumulated counts. The capacity of' the counter was 1,24 counts, and when capacity was reached the output signal returned to zero. This output signal was an accurate integration of' the counts-per-minute curve from the ratemeter and was fed to the second channel of the strip chart recorder. Specific activity of tryptophan in protein hydrolysate samples was calculated by using a factor determined from measurements of standard amino acid mixtures containing known amounts of' ["C]tryptophan. E- 14, z 12, 2!o,., 8 4 a 6 2,-T RESULTS Experiments with mixed cultures of ruminal microbes. The rate of incorporation of' radioactivity from [2-"CIIAA (2.4 x 1-' M, 8 Ci/mol) by microbial cells in ruminal fluid from a sheep is shown in Fig. 1. Addition of tryptophan to the fermentation mixture and dilution of' the specific activity of the [1-14C]IAA by addition of unlabeled IAA greatly reduced the rate of incorporation of' radioactivity into cells. Addition of substrate (ground alfalfa) increased the rate of incorporation of' 4C into cells, presumably due to stimulation of the growth rate of IAA-utilizing microorganisms. The distribution of' "C in fractions obtained from microbial cells after incubation of ruminal contents with [1-"C]IAA is given in Table 1. With ruminal fluid from the sheep, most of' the "C in washed microbial cells was in the protein fraction that remained after treatment with hot 5% (wt/vol) trichloroacetic acid. The ruminal ingesta from the cow shows a similar result, but the differential centrifugation produced a proto- cr4-4< GROUND ALFALFA (.5g/mI) NONE TRYPTOPHAN (13 M) NINDOLE ACETIC ACID (IO3M) MINUTES FIG. 1. Incorporation of 14C from [-_4CJIAA into microbial cells during in vitro fermentation of strained ruminal contents from a sheep. Materials indicated were added to the culture at 2 min.

3 VOL. 117, 1974 TRYPTOPHAN BIOSYNTHESIS BY RUMEN BACTERIA TABLE 1. Distribution of radioactivity after incubation of ruminal material with [1-"C]indole-3-acetic acid under CO2 at 39 C for 12 min Substance Radioac aoc - tivity Of ruminal fluid Radioactivity of whole ruminal ingesta from a cowa from a 3 x 25,Ox sheepa g ppt5 g ppt Fractions of cells Cold trichloroacetic acid extract Ethanol-ether extract acid extract Wash of hot trichloroacetic acid precipitate acid precipitate a Units are x 13 disintegrations per minute. For whole cultures of a sheep and cow, radioactivity was 2.34 and 17.5 x 1' dpm, respectively. b Centrifugation at 3 x g for 5 min precipitated most of the protozoa as well as many bacteria. zoa-free bacterial precipitate that contained considerable radioactivity in the protein fraction. Most of the '4C in hydrolysates of this fraction migrated with tryptophan (detected with Erlich reagent) during paper chromatography on all of the chromatographic systems used. The R, of the radioactive substance and of known tryptophan with the butanol-acetic acid solvent system was.5, whereas that of [1- "4C]IAA was.98. With the collidine system buffered at ph 9, tryptophan (R,.59 to.63) migrated farther than other amino acids in the hydrolysate. Experiments with pure cultures. Pure cultures of rumen bacteria cultured in media containing [1-'4C ]IAA included: Bacteroides ruminicola strains 23 and GA33, Eubacterium ruminantium strain B-4, Peptostreptococcus elsdenii [Megasphaera elsdenii](29) strain B-159, Butyrivibrio fibrisolvens strain D-1, Ruminococcus flavefaciens strain C-94, R. albus strains 7, 2, B3 37, and D-89, and the nonruminal anaerobe R. bromii ATCC With the exception of the R. albus cultures, these organisms incorporated less than 1% of the "4C from the culture medium. All four of the R. albus strains tested readily incorporated '4C from [1-"C]IAA, and in Table 2 distribution of radioactivity in cell fractions is given. We have not attempted to explain the differences between cultures shown in Table 2. Less than 2% of the "C in culture supernatant fractions was present as "CO2. Most of the "C in cells was in the hot trichloroacetic acid precipitate, protein fraction. Labeled compounds in the other fractions were not identified. Tryptophan was the only labeled amino acid detected when protein hydrolysates were examined by preparing radioautographs of paper and thin-layer chromatograms. When amino acids from protein hydrolyzed with alkali were separated on the cellulose thin-layer system, two spots containing "4C that corresponded with known D- and L-tryptophan were detected. A single radioactive spot corresponding with L-tryptophan was found when the protein was hydrolyzed with p-toluenesulfonic acid. When hydrolysate from R. albus strains (Tables 2 and 3) were analyzed on the amino acid analyzer-anthracene flow cell system, a single peak of radioactivity that corresponded to tryptophan was detected. When unlabeled IAA was added to culture medium containing [1-4C ]IAA, the reduction in incorporation of radioactivity was roughly proportional to the change in specific activity of the IAA (Table 3). The ratios of the molar specific activities of tryptophan (measured) to IAA (calculated, based upon dilutions of a TABLE 2. Incorporation of radioactivity from [1-14CJIAA by growing cultures of Ruminococcus albus Substance 177 Incorporation of radioactivitya by strains: 2' 7c B,37 d D89e Whole culture... 39,4 22,3 6,8 6,56 Cells... 3,62 2,89 2,65 2,2 Fractions of cells Cold trichloroacetic acid extract Ethanol-ether extract acid extract Wash of hot trichloroacetic acid precipitate acid precipitate 3, 2,18 2,46 1,71 a Units are x 13 disintegrations per minute per 2 ml of culture and fractions thereof. I Strain 2 grown to an optical density (OD) of.68 (15 h) and IAA = 8 Ci/mol. c Strain 7 grown to an OD of.8 (16 h) and IAA = 4.23 Ci/mol. d Strain B337 grown to an OD of.85 (17 h) and IAA = 4.23 Ci/mol. Strain D89 grown to an OD of.9 (2 h) and IAA = 4.23 Ci/mol.

4 178 ALLISON, ROBINSON, AND BAETZ J. BACTERIOL. TABLE 3. Effect of concentration and specific activity of [1-4C]-IAA upon incorporation of 14C into growing cultures of R. albus strain 2 Sp act of: IAA incor- Cultures - Protein porated ICAuAa cwhle cellsc fraction Try ptno-ito ha of cellsc cellse Iculture' A 2,9 35,8 3,84 3,58 1, B 82 34, 1, C 43 34, a Measurements are calculated disintegrations per minute per nanomole of IAA. The concentrations of IAA were: 1.38, 4.36, and 8.37 x 1-4 M for cultures A, B, and C, respectively. b Units are x 13 disintegrations per minute per 95 ml of culture medium. c Units are x 13 disintegrations per minute. Cells were harvested after 23 h at an optical density (OD) of.62,.77, and.81 for cultures A, B, and C, respectively. Measurements of cellular "4C in A and B have been corrected to that expected for incorporation into cells grown to an OD of.81. d Units are disintegrations per minute per nanomole of tryptophan measured by the amino acid analyzer flow cell counting system. eiaa incorporated per 95 ml of culture (mmol). Corrected for differences in growth as in c. weighed quantity of IAA) ranged from.62 to.74. The tryptophan content of the cell protein fraction from the R. albus strain 2 culture was estimated to be 1.2 g/1 g of protein when the p-toluenesult'onic acid hydrolysate was analyzed with the amino acid analyzer. R. albus strain 2 was grown in media containing [14C ]bicarbonate (approximately 12 ACi/5 ml) with and without added unlabeled IAA (2 x 1-' M). The protein fractions from the cells in each culture contained approximately 1.6 ACi of the '4C. Radioactivity was found in most amino acids but was not detected in leucine or histidine, and relatively small quantities were found in isoleucine and valine. The ratio of the measured molar specific activity of tryptophan to that of carbonate recovered from the medium after growth of the cultures was 1.95 and 1.9 for cultures grown with and without added IAA, respectively. These calculations of' the specific activity of' tryptophan are based upon a single measurement from each culture, and the precision of the determinations is not known. R. albus strain 7 was cultured in a medium containing [3- "C ]serine (4 gci/2 ml, 3.8 x 1' M). The protein fraction (132, dpm) was hydrolyzed with alkali and chromatographed on one- and two-dimensional paper chromatography systems and with the amino acid analyzer-anthracene flow cell system. Radioautographs of the paper chromatograms were prepared, and most of the "C migrated with serine; some was present in the alanine area, but none was detected in the tryptophan area. These findings were confirmed with the amino acid analyzer system. When R. albus strain 7 was cultured in medium containing DL-shikimic acid (1 uci/2 ml, 5.8 x 1-5 M), the protein fraction of the cells contained less than 6, dpm. No attempt was made to identify labeled compounds. DISCUSSION Experiments were designed to test the hypothesis that ruminal microbes are capable of carboxylating IAA to synthesize the carbon skeleton of tryptophan. We found the following. (i) When ruminal ingesta from a sheep was incubated with [1-`4C ]IAA, radioactivity was incorporated into microbial cells. (ii) The incorporation of labeled IAA was decreased when either unlabeled IAA or tryptophan was added to the incubation mixture, but was increased when substrate (ground alfalfa) was added. (iii) Most of' the "4C in microbial cells was in the protein fraction. (iv) When ruminal contents from a cow were incubated in a similar manner and protozoal cells were removed by differential centrif'ugation. the remaining bacterial cells contained "C, and most of' this was in the protein fraction of the cells. (v) Tryptophan was the only amino acid in hydrolysates of these protein fractions that contained a significant quantity of "4C. (vi) Of' the pure cultures of ruminal bacteria tested, R. albus cultures were the only strains that incorporated and converted IAA into tryptophan, and all four strains of R. albus studied were able to make this conversion. Evidence for the identity of tryptophan as the labeled substance in the protein fractions was obtained by using paper, thin-layer, and column chromatographic procedures with samples hydrolyzed by alkali as well as with p-toluenesulfonic acid. Since tryptophan is racemized during alkaline hydrolysis, the evidence for production of labeled D-tryptophan during alkaline hydrolysis of protein, but not during acid hydrolysis, supports the other evidence concerning the identity of the labeled product in protein. When R. albus was grown in media containing IAA ranging from 1.38 to 8.37 x 1' M, similar quantities of IAA were incorporated (Table 3). Thus, the lower concentration must saturate the incorporation or utilization reac-

5 VOL. 117, 1974 TRYPTOPHAN BIOSYNTHESIS BY RUMEN BACTERIA tions. No attempt was made to compare incorporation rates at lower concentrations of IAA, but with Pseudomonas savastanoi the IAA transport system was saturated near 5 x 1-5 M (22). Comparison of the specific activities of IAA and tryptophan from cellular protein from R. albus (Table 3) indicates that most of the tryptophan was synthesized from IAA. The mechanism for tryptophan biosynthesis from IAA is not known. We propose, however, that this proceeds via a reductive carboxylation reaction to synthesize indole pyruvate which is then available for transamination. This proposal is based upon evidence for analogous reductive carboxylations by anaerobic bacteria to synthesize amino acid carbon skeletons (1, 5). The involvement of ferredoxin in certain of these carboxylations has been demonstrated (3, 9, 13). The position of 14C in tryptophan synthesized from [1-14C]IAA has not been determined. If the reactions proceed as suggested above, one would expect '4C to be in carbon number 2. Since the radioactivity from [1-14C]IAA was found mainly in tryptophan, it seems likely that the IAA molecule was not catabolized and that the intact IAA molecule was utilized for tryptophan biosynthesis. The position of "4C in tryptophan from cultures grown in "4CO2 is also unknown, but if the proposed pathway operates one would find the label in the carboxyl carbon. Since R. albus produces C2, the specific activity of bicarbonate in the medium would decline during growth of the culture. This might explain partially the finding that the specific activity of tryptophan synthesized was higher than that of the carbonate in the culture medium after growth. The presence or absence of nonradioactive IAA in the growth medium had little, if any, effect upon the specific activity of the tryptophan synthesized in the presence of "CO2. If IAA had exerted an effect on incorporation of "CO2, one might interpret the results to mean that tryptophan was synthesized by a mechanism that does not involve IAA when IAA was not supplied. In the absence of an effect, however, one cannot exclude the possible function of some other biosynthetic mechanism. Studies with tryptophan-synthesizing systems and tryptophan synthetase from Escherichia coli and Neurospora crassa have shown that carbons 1, 2, and 3 of tryptophan arise from serine (35). Tryptophanase from E. coli is also capable of tryptophan synthesis from serine, cysteine, or S-methyl cysteine in the presence of indole (25). Indole acetic acid is not an intermediate in these reactions. Although we were unable to demonstrate incorporation of "C from serine or shikimic acid into tryptophan by R. albus, the available evidence is not enough to eliminate conclusively the possible function of known pathways in addition to the mechanism involving IAA. Tryptophan is one of the amino acids that is relatively slowly metabolized by ruminal microbes (1, 17, 31). Among the products of ruminal metabolism of tryptophan are IAA, 3-methyl indole, and indole (11, 16, 18, 3). The organisms responsible for tryptophan catabolism are not known. Recently Carlson and co-workers (11) reported that 3-methyl indole is the major product of tryptophan and IAA metabolism by mixed ruminal microbes in vivo and in vitro. The concentrations of IAA and tryptophan added in most of these experiments were considerably higher than the ruminal concentrations expected in animals on usual rations. It may be that the rate of metabolism, the metabolic pathway used, and the end products formed are influenced by the concentration of tryptophan added to the mixed culture fermentation. A concentration dependent regulation of degradative pathways has been demonstrated for ruminal fermentation of glycine (34). Although R. albus is an important part of the microbial population in the rumen of cattle and sheep (14, 15), the experiments reported here do not permit an estimation of the relative importance of tryptophan biosynthesis from IAA. Many rumen bacteria have not been tested for this biosynthetic capacity, but of those tested here only R. albus used IAA. It thus seems probable that most of the tryptophan synthesized by ruminal microbes is synthesized by some other pathway. The ability of R. albus to use IAA for tryptophan biosynthesis may be another example of selection of a microbe to fit an ecological niche. It would not be surprising to find IAA-utilizing anaerobes, including R. albus, in other gastrointestinal environments that contain IAA (33). We tested R. bromii for this reaction because it is similar to R. albus and is one of the predominant species in fecal samples from humans and swine (24). Since we were unable to grow R. bromii in the defined medium which did not contain tryptophan, we are uncertain concerning its ability to use IAA. ACKNOWLEDGMENTS 179 We are grateful for the technical assistance of Jerry A. Bucklin. R. W. Dougherty prepared animals with ruminal cannulas, and J. L. Riley designed and constructed the electronic counting circuit used with the amino acid analyzer.

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