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The distribution of free amino acids in the contents of various regions of the gastrointestinal tract (stomach, upper small intestine, lower small intestine, cecum, upper colon and lower colon) was studied in germfree and conventionalized mice. Particular emphasis was placed on the conversion of tryptophan to indole as a probe for studying intermicrobial interactions and microbe-host interactions in vivo. Great differences were observed in the free amino acid content of the various regions of the digestive tract in each type of mouse and also in any one region between germfree and conventionalized mice. As would be expected, there were fewer differences in amino acid distribution between the types of mice in both regions of the small intestine. This correlates with a much lower population of microorganisms in these regions. The changes in free amino acid content and distribution produced by microflora are great enough to serve as a good probe for studying the interactions of a limited number of species of microbes in gnotobiotic animals and assign possible specific functions to each species.

A crystalline tryptophanase can be obtained from extracts of Spaerophorus funduliformis using a heat treatment, hydroxyapatite chromatography and solubility in solutions of (NH4)2SO4 as a function of pH and temperature. The purified enzyme is homogeneous by several criteria. S. funduliformis tryptophanase has a specific activity of 11.5-13.5 and requires pyridoxal 5'-phosphate for enzymatic activity. Like other tryptophanases that have been studied, the S. funduliformis enzyme is a tetramer protein consisting of four apparently identical subunits. The native enzyme has a sedimentation coefficient of 11.2 S and a molecular weight of 244 000. In solutions of 5 M guanidine - HCl, 8 M urea, and sodium dodecylsulfate, at high pH or in the presence of thiols, the enzyme dissociates to 59 000 molecular weight species which are homogeneous by the criterion of weight. Peptide maps of the reduced holo-tryptophanase show one pyridoxal-containing peptide and, lacking agreement with the determined amino acid composition, suggest that the subunits of the enzyme contain a high degree of internal sequence homology.

Bacillus alvei apotryptophanase readily dissociates at low protein concentration and sediments at 5.7 S (dimer) in 0.01 M potassium phosphate (pH 7.8) from 9 to 33 degrees. With temperature held constant at 9 degrees, increasing the potassium, sodium, or ammonium phosphate buffer concentration increases the sedimentation value to 8.0 S. Increasing the monovalent cation concentration alone does not have the effect. Imidazole and pyridoxal compete with phosphate, preventing the effect. Raising the temperature to 26 degrees in the presence of high concentrations of potassium phosphate increases the sedimentation constant to 9.4 S. The addition of pyridoxal-P converts the dimer to a 9.4S tetramer. The conversion is dependent upon coenzyme concentration, temperature, and the nature of monovalent cation present. The Km for pyridoxal-P for the sodium form of the enzyme is more than tenfold greater than the Km for the potassium form of the enzyme. 2'-Methyl, 2'-hydroxyl, 6-methyl, and the N-oxide of pyridoxal-P are active in the association of dimer to tetramer but to differing extents. Analogs altered in the 4'-formyl position are also inactive structurally. Anthranilic acid, a competitive inhibitor of tryptophan, and 8-anilino-1-naphthalenesulfonic acid (ANS), a competitive inhibitor of pyridoxal-P binding, are both active in affecting the dimer to tetramer association but tryptophan is not. The dimer and tetramer are spectrally distinguishable through circular dichroic measurements, fluroescence quenching with pyridoxal-P or pyridoxal, and fluorescence enhancement with ANS. Pyridoxal-P causes the release of ANS from an ANS-apoenzyme complex.

The conditions for synthesis, purification, and properties of tryptophanase by a marine organism (Vibrio K-7) were studied. Tryptophanase was induced by tryptophan and its analogs, and partially repressed by 0.5% glucose or glycerol. NaCl (0.4 M) was required for optimal growth and tryptophanase activity in whole cells. The enzyme was purified to 92% homogeneity by heat treatment, hydroxyapatite chromatography and fractionation with ammonium sulfate. This tryptophanase has been found to have kinetic properties similar to the tryptophanase from other microorganisms. It carries out both alpha, beta-elimination reactions (using tryptophan, serine, cysteine and S-methylcysteine as substrates) and beta-replacement reactions (forming tryptophan from indole and serine, cysteine or S-methyl-cysteine). The enzyme has a sedimentation coefficient of 9.2S and requires pyridoxal 5'-phosphate as a cofactor. The optimal pH for the tryptophanase reaction is pH 8.0.

Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.

The activity of the enzyme tryptophanase in the enteric environment was investigated to elucidate the significance of the enzyme in the metabolism of Escherichia coli. The tryptophanase activity, tryptophan content, and indole concentration as well as the numbers of E. coli were determined in the intestinal and fecal contents of conventional, germ-free, and monocontaminated axenic laboratory mice. Increasing the tryptophan content of the diet of mice having a conventional microflora increased the tryptophanase activity of the enteric microflora by a factor of almost 2 but did not increase the numbers of E. coli either absolutely or relative to other facultative enteric coliforms. In the enteric environment, E. coli is responsible for very little tryptophanase activity, a fraction calculated to be less than 0.02%. The values for the experimental parameters were much the same in the contents of the cecum and in the fecal material.

A partially purified enzyme (120-fold) from Leuconostoc mesenteroides catalyzed the reversible N-acetylation of d-glucosamine-6-phosphate. Coenzyme A was not required and inhibited the reaction rate. Neither d-glucosamine nor N-acetyl-d-glucosamine served as a substrate for the reversible reaction. The enzyme preparation retained 50% of its original activity after 5 min at 100 C. The K(m) for acetate was 7.7 x 10(-2)m in the presence of 2 x 10(-2)md-glucosamine-6-phosphate. The K(m) for d-glucosamine-6-phosphate was 5.0 x 10(-3)m in the presence of 0.64 m acetate. The product of the reaction was characterized by comparison with N-acetyl-d-glucosamine-6-phosphate prepared by enzymatic phosphorylation of N-acetyl-d-glusamine. The characterization tests were: chromatographic migration, acid hydrolysis, enzymatic dephosphorylation, sodium borohydride reduction, and periodate oxidation. The equilibrium constant for the reaction was about 7.5 m for the expression K = (d-glucosamine-6-phosphate)(acetate)/N-acetyl-d-glucosamine-6-phosphate. The standard free energy of the reaction was approximately 1,200 cal per mole.

The distribution of tryptophanase was studied. The highest observed specific activity, mumoles per minute per milligram (dry weight) cells, is given in parentheses after each species. Tryptophanase was inducible and repressible in Escherichia coli (.914), Paracolobactrum coliforme (.210), Proteus vulgaris (.146), Aeromonas liquefaciens (.030), Photobacterium harveyi (.035), Sphaerophorus varius (.021), Bacteroides sp. (.048), and Corynebacterium acnes (.042). The enzyme was constitutive and nonrepressible in Bacillus alvei (.013), and was inducible but not repressible by glucose in Micrococcus aerogenes (.036). Indole-positive bacteria were found in fecal or intestinal samples from a variety of animals among the mammals, reptiles, insects, molluscs, fish, crustaceans, and amphibians.

Dobrogosz, Walter J. (University of Illinois, Urbana) and Ralph D. DeMoss. Pentose utilization by Pediococcus pentosaceus. J. Bacteriol. 85:1356-1364. 1963.-Data are presented which indicate that pentoses are metabolized by Pediococcus pentosaceus through the use of an inducible phosphoketolase pathway. The utilization of each of the pentoses (l-arabinose, d-ribose, and d-xylose) appears to involve a different regulatory process. Thus, d-ribose was fermented by cells grown on any pentose or glucose, although growth on a pentose resulted in an increased ribose-fermenting capacity. l-Arabinose fermentation was only observed when cultures were grown in the presence of this pentose. Similarly, d-xylose fermentation was a specific response to the presence of xylose in the growth medium. In this case, however, growth occurred only at a very low rate unless another readily utilizable substrate (in low concentration) was added to the growth medium. Under these conditions, an aerobic atmosphere was more stimulatory than anaerobiosis for induction and growth on xylose. The possible cryptic nature of xylose utilization in this organism is described.

Bacillus alvei excretes indole during early exponential growth in acid-hydrolyzed casein medium. l-Threonine is the amino acid responsible for "early" indole excretion, and the amount of indole excreted is directly related to the amount of l-threonine in the medium. "Early-indole" excretion can be prevented by the continuous addition of serine (3.1 mumoles per ml per hr) or by substituting a mutant with an impaired ability to degrade serine. The addition of serine to a culture during the period of indole excretion halts the excretion and stimulates indole utilization. Threonine is a competitive inhibitor of serine (K(i) = 0.6 m) in the tryptophan synthetase B reaction. The internal tryptophan concentration increases during the period of indole excretion, suggesting that threonine acts by increasing the activity of the tryptophan pathway. This view is supported by experiments demonstrating that anthranilic acid and indoleacrylic acid also stimulate indole excretion. A metabolic explanation is offered and discussed.

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei. J. Bacteriol. 91:667-672. 1966.-Indole excretion occurred early in the exponential growth phase, and derived mainly from biosynthetic intermediates of tryptophan. Tryptophan cleavage by tryptophanase contributed about 1.5% of the indole excreted. In the presence of exogenous tryptophan (5 to 10 mug/ml), excretion of early indole was not observed. Experiments with isotopically labeled indole and tryptophan showed that a low rate of endogenous tryptophan biosynthesis occurred constantly during growth. Both exogenously and endogenously supplied tryptophan were degraded by tryptophanase. As a consequence, the intracellular tryptophan concentration appeared to be maintained at a constant low level. It was suggested that the action of tryptophanase is an example of an enzymatic mechanism which controls the level of a specific metabolite pool.

Enzyme extracts of Leuconostoc mesenteroides were found to contain at least four separate kinases: one active with glucose, glucosamine, and N-acetylglucosamine; one active with fructose and mannose; and two active with gluconate, one constitutive and one inducible. The molecular sizes of all the kinases, estimated from sucrose gradient centrifugation, are about the same, 37,000 to 50,000 daltons, except the constitutive gluconate kinase, which is about 100,000 daltons. Apparent Michaelis constants were calculated for all of the substrates mentioned. The kinases are separable on triethylaminoethyl cellulose.

Anthranilate synthetase from Bacillus alvei was partially purified by ammonium sulfate fractionation and was stabilized by glycerol. The reaction mechanism of the enzyme was found to be sequential with respect to substrate, and the enzyme formed a hydroxamic acid in the absence of Mg(++). The K(m) for chorismic acid was 1.25 x 10(-4)m, and the K(m) for l-glutamine was 5.5 x 10(-4)m. Enzyme activity was inhibited by tryptophan noncompetitively with respect to chorismic acid and uncompetitively with respect to l-glutamine. An analysis of the inhibition patterns indicated that tryptophan may act as a dead end inhibitor and bind at the catalytic site. Enzyme activity could be completely inhibited in vitro and in vivo under the appropriate conditions, and enzyme synthesis was sensitive to repression by tryptophan. A sedimentation coefficient of 5.5S and an estimated molecular weight of 90,000 were obtained for the enzyme.

Raps, Shirley (University of Illinois, Urbana) and R. D. DeMoss. Glycolytic enzymes in Zymomonas mobilis. J. Bacteriol. 84:115-118. 1962-An enzyme extract of Zymomonas mobilis (Pseudomonas lindneri) was capable of fermenting glucose-6-phosphate to CO(2) and ethanol. The extract was found to contain phosphohexoisomerase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, but no demonstrable phosphohexokinase. The lack of isotope-mixing found in earlier studies is, thus, explained on an enzymatic basis.

Tryptophanase from Bacillus alvei exhibited the expected spectrum of pyridoxal-5'-phosphate-dependent reactions. It exhibited l-serine dehydratase, S-alkyl-cysteine lyase, and cysteine desulfhydrase activities, as well as the classic tryptophanase reactions (all beta elimination reactions). It also acted as a tryptophan synthetase (beta replacement reactions) using indole plus l-serine or l-cysteine or S-methyl-cysteine as substrates. The beta elimination reactions are simple competitors of the replacement reactions for the same amino acid substrates. The kinetics of the reactions were examined in detail using a coupled continuous spectrophotometric assay. A product (indole) inhibition study of the beta elimination reaction with tryptophan showed simple, noncompetitive inhibition; the same study with allosubstrates showed noncompetitive inhibition by indole. These product studies provided data on the beta replacement reactions as well. The results are discussed in terms of a mechanism for the B. alvei tryptophanase.

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.

The prevalence of indole-positive organisms in the gut and environment of marine animals was studied. Indole formation by a group of the isolates was found to occur only in the presence of tryptophan. The isolates examined were all assigned to the genus Vibrio.

Dobrogosz, Walter J. (University of Illinois, Urbana) and Ralph D. DeMoss. Induction and repression of l-arabinose isomerase in Pediococcus pentosaceus. J. Bacteriol. 85:1350-1355. 1963.-The inducible l-arabinose isomerase of Pediococcus pentosaceus can be rapidly and conveniently measured in whole-cell preparations by use of a standard colorimetric procedure originally developed for studies with cell-free enzyme preparations. The enzyme is measured by its ability to catalyze the isomerization of l-arabinose to l-ribulose. Whole cells suspended in a suitable buffer and pretreated with toluene were shown to exhibit this isomerase activity at a level comparable with that observed in cell-free enzyme preparations. Conditions for optimal induction of l-arabinose isomerase are described. In addition, it was determined that the formation of this enzyme is subject to repression by glucose, i.e., via catabolite repression.

Unger, Leon (University of Illinois, Urbana), and R. D. DeMoss. Metabolism of a proline analogue, l-thiazolidine-4-carboxylic acid, by Escherichia coli. J. Bacteriol. 91:1564-1569. 1966.-Resting cells of Escherichia coli K-12, pregrown in a proline- and thioproline-free medium, oxidize the proline analogue, l-thiazolidine-4-carboxylic acid (l-thioproline), without a lag with the consumption of 1 atom of oxygen per mole of thioproline. The organism also oxidizes cysteine and formaldehyde, the chemical precursors of thioproline. The total oxygen consumed is the same whether the substrate is thioproline, cysteine, formaldehyde, or an equimolar mixture of cysteine and formaldehyde. The results suggest that neither cysteine nor formaldehyde are free intermediates in the oxidative pathway. Thioproline is available as a metabolic carbon source for the synthesis of the ribonucleic acid bases, guanine and uracil.

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological effects of a constitutive tryptophanase in Bacillus alvei. J. Bacteriol. 90:604-610. 1965.-Tryptophanase synthesis in B. alvei is not under the control of tryptophan and is not subject to catabolite repression. Exogenously supplied tryptophan was converted to indole by tryptophanase, and was excreted into the culture medium. The amount of indole excreted was dependent upon the concentration of tryptophan supplied. At intermediate levels of tryptophan (5 to 15 mug/ml), the excreted indole was completely reutilized by the cell, in contrast to the result with higher levels. Indole reutil zation was shown to be dependent upon a functional tryptophan synthetase. In the absience of exogenous tryptophan, indole was excreted into the culture medium at an earlier physiological age. The early indole was shown not to be a consequence of tryptophanase action. The early indole accompanied uniformly the normal process of tryptophan biosynthesis, and the fission of indole-3-glycerol phosphate was suggested as the origin of the excreted indole.

Unger, Leon (University of Illinois, Urbana), and R. D. DeMoss. Action of a proline analogue, l-thiazolidine-4-carboxylic acid, in Escherichia coli. J. Bacteriol. 91:1556-1563. 1966.-The effect of the proline analogue, l-thiazolidine-4-carboxylic acid (thioproline), on growth, and its relation to the metabolic function of proline in protein synthesis in Escherichia coli K-12, has been studied. Thioproline causes linear growth in E. coli within one generation. The inhibition is specifically reversed by the simultaneous addition of l-proline. Thioproline, or a closely related metabolic derivative, is incorporated into bacterial proteins. Proline antagonizes the incorporation of "thioproline" into protein. The analogue specifically inhibits the rate and extent of prolyl-ribonucleic acid formation. The effectiveness of thioproline as a proline analogue is attributed to its ability to interfere with the utilization of proline for protein synthesis and to mimic proline in its function of being incorporated into proteins. The effect of the incorporation of thioproline on protein structure and enzyme activity is discussed.