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The purpose of this essay is threefold: to give an outline of the life and the various achievements of Theodor Escherich, to provide a background to his discovery of what he called Bacterium coli commune (now Escherichia coli), and to indicate the enormous impact of studies with this organism, long before it became the cornerstone of research in bacteriology and in molecular biology.

The career of Theodor Escherich (1857-1911) qualifies him as the first pediatric infectious diseases physician. His landmark bacteriologic studies identified the common colon bacillus (now known as Escherichia coli), and he was very committed to pediatrics, serving as chairman of several prominent departments of pediatrics, including the Department of Pediatrics at the University of Vienna and St. Anna's Children's Hospital in Vienna, Austria, arguably Europe's most prestigious pediatric position.

New ideas in science frequently arise from neglected or distorted antecedents. This essay deals with the idea of biochemical unity, encapsulated in Jacques Monod's well-known phrase, dating from 1954: "Anything found to be true of E. coli must also be true of elephants." An earlier version of this phrase,--"From the elephant to butyric acid bacterium--it is all the same!"--was coined in 1926 by the Dutch microbiologist Albert Jan Kluyver. In that year Kluyver and his associate Hendrick Jean Louis Donker published a celebrated paper, "Unity in Biochemistry." The concept of biochemical unity had many antecedents, but these had never caught on. The Kluyver-Donker paper has often been regarded to provide a boost to biochemical and especially to microbiological thinking. Its interpretations and misinterpretations represent an encapsulated history of biochemistry. The present paper examines the history of the concept of biochemical unity from before to beyond Kluyver, investigates the two "elephant" phrases and their possible relationships, and ends with a discussion of the attractiveness of unifying ideas in science.

Two blue bile pigments were formed under anaerobic conditions from the tetrapyrrole precursor delta-aminolevulinate by cells and cell extracts of Clostridium tetanomorphum. These compounds were also formed by cell extracts from the octacarboxylic tetrapyrrole, uroporphyrin III. Bactobilin, the first bacterial bile pigment to be discovered, is related to uroporphyrin I. The present results hence increase the number of bile pigments related to bactobilin. Bactobilin and its isomers differ markedly from the eukaryotic bile pigments which are all related to the dicarboxylic compound, protoporphyrin IX. The enzyme participating in the formation of the bacterial bile pigments was obligatorily anaerobic, in decided contrast to the only other known bile pigment-forming enzyme, the eukaryotic oxygen-requiring heme oxygenase.

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.

Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reverse-phase chromatography on cartridges containing a C18 spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis.

L-Alanine:4,5-dioxovalerate aminotransferase, the enzyme that catalyzes the transamination between alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from extracts Clostridium tetanomorphum by acetone precipitation and successive tetanomorphum by acetone precipitation and successive chromatography on Sephadex G-150, hydroxyapatite, Octyl-Sepharose, and SP-Sephadex C-50. The enzyme is pure by the criterion of disc gel electrophoresis with varying polyacrylamide concentrations. It is dimeric, and has an apparent molecular weight of 111,000. Each molecule contains 2 molecules of pyridoxal 5-phosphate. The apparent Km values for 4,5-dioxovalerate and L-alanine are 0.26 and 1.96 mM, respectively. In addition to alanine, glutamate also is an effective amino group donor. The enzyme is inhibited by various keto acids as well as by inhibitors of pyridoxal phosphate-containing enzymes. It was possible to show that 4,5-dioxovalerate is formed by cultures of C. tetanomorphum when grown in the presence of 0.2 M levulinate, an inhibitor of 5-aminolevulinate dehydratase.

When a solution of 14C-labeled L-threonine is incubated with aquocobalamin in the presence of excess of a reductant such as 2-mercaptoethanol or sodium borohydride, a labeled corrinoid adduct is formed. The corrinoid participates stoichiometrically, and not catalytically. Since 35S-labeled 2-mercaptoethanol is not part of the adduct, the reduction of the corrinoid from the Co(III) to the Co(II) form is a prerequisite for the reaction. Various independent tests show the formation of isopropanolamine. The reaction system is an attractive model for the so far unexplained decarboxylation of threonine to isopropanolamine in the course of corrinoid biosynthesis.

A method for partial purification of an enzyme system from greening wheat leaves which converts 14C-labeled glutamate to delta-aminolevulinic acid is described. The purification entails the successive use of anion and cation exchange, followed by molecular sieving. The enzyme system is unstable in crude form, but the stability is markedly increased after column chromatography on CM-cellulose. The pH profile and the cofactor requirements suggest that at least two enzymes are involved.

A blue bile pigment, possessing four acetic and four propionic acid side chains has been isolated from extracts of the anaerobic microorganism Clostridium tetanomorphum and in smaller amounts from Propionibacterium shermanii. The compound could be prepared in larger amounts by incubation of C. tetanomorphum enzyme extracts with added delta-aminolevulinic acid. The ultraviolet-visible, infrared, and proton magnetic resonance spectra of the pigment indicate a chromophore of the biliverdin type. Field-desorption mass spectrometry of the purified methyl ester showed a strong molecular ion at m/e = 962. This corresponds to the molecular weight expected for the octamethyl ester of a bilatriene type of bile pigment structurally derived from uroporphyrin III or I. Of the five possible structures, two could be eliminated by proton magnetic resonance spectroscopy. The name bactobilin is proposed for this previously unreported bile pigment.