Various investigators1 2, 3 have shown that pyridoxine (vitamin B6) under certain conditions is a necessary growth factor for certain species of lactic acid bacteria. One such organism is Streptococcus lactis R. In a detailed study of the requirement of this organism for pyridoxine, Snell, et al.,4 showed that there existed in natural products a substance which could be derived from pyridoxine by metabolic processes, which far surpasses pyridoxine in its physiological activity for this organism. To distinguish this substance from synthetic pyridoxine it was tentatively called "pseudopyridoxine." It was later shown5 that if heat sterilization were avoided, pyridoxine was almost without growth-promoting activity for this organism. It became highly active on autoclaving with the basal culture medium. The degree of activity increased as the period of heating was increased. The effect was traced to interaction during autoclaving between pyridoxine and the amino acids of the medium; presumably "pseudopyridoxine" or a substance of similarly high activity for the test organism was formed in small amounts.While investigating the effect of individual amino acids in promoting the above "activation" of pyridoxine during heating, it was found that large amounts of alanine completely replaced pyridoxine as a growth factor for this organism. Glycine, on the other hand, inhibited growth. This inhibitory effect of glycine was removed, within limits, by additional amounts of pyridoxine, but not by additional amounts of any of the other vitamins. It was similarly removed-even more effectively-by alanine, 66 PROC. N. A. S.

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Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by a-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.Inducible histidine decarboxylases (HDCs) of two types are known. The purified enzymes from four different grampositive organisms of three different genera have all proved to be oligomeric pyruvoyl enzymes (14) that differ in sequence but that contain two different types of subunits derived by an unusual process from a pyruvoylfree, enzymatically inactive proenzyme subunit (6,11,12,14). In contrast, HDC from a gram-negative bacterium, Morganella morganii, contained no pyruvoyl group, but required pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme (18). Bacteria of both types have been implicated in histamine poisoning in humans: M. morganii in fish products (1) and Lactobacillus buchneri in cheese (23).HDC from M. morganii is the only pyridoxal-P-dependent HDC that has been extensively studied in pure form (18). It is a tetramer of identical subunits (18) of known amino acid sequence (20). To extend this comparison, we describe here the purification and comparative properties of HDCs from two additional gram-negative bacteria, Klebsiella planticola and Enterobacter aerogenes. Like the enzyme from M. morganii, both enzymes proved to be homomeric and to require pyridoxal-P as a coenzyme. These results increase our knowledge of these important decarboxylases and also extend the correlation between Gram staining properties and the type of HDC produced. MATERIALS AND METHODSCultures and growth conditions. Cultures of M. morganii ATCC 35200, K. planticola ATCC 43176, and E. aerogenes ATCC 43175 were maintained by monthly transfer on brain infusion agar slants (K. pneumoniae and E. aerogenes were originally obtained from Stephen Taylor as strains T2 ST-38 and ST-40, respectively, and were sent to the American Type Culture Collection, Rockville, Md., with his permission). For enzyme production, cultures were grown at 30°C for 24 h without agitation in a hydrolyzed casein-yeast * Corresponding author.extract-glucose-histidine medium (18) from a 10%...

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