An obligately symbiotic bacterium, strain T, associating with a specific strain S of a thermophilic Bacillus sp. was isolated during screening for micro-organisms that produce heatstable tryptophanase and P-tyrosinase. A colony containing both strains was finally isolated by successive selection with bacitracin. In mi,xed culture with Bacillus strain S under low aeration conditions at 60 "C, growth of strain T was initiated following lysis of the Bacillus cells. No independent growth of strain T was observed in any of the media tested, even in the presence of other thermophilic Bacillus strains or cell-free extracts of strain S or its killed cells. N o physical adhesion between cells of strains S and T was observed microscopically. Tryptophanase and Ptyrosinase production by strain T were induced by L-tryptophan and L-tyrosine, respectively. The name Symbiobacterium thermophilum gen. nov., sp. nov. is proposed for strain T which is a Gram-negative rod (0.25-0.35 pm diameter) with a multilayer surface structure containing meso-diaminopimelic acid. The G + C content of the DNA is 65.1 mol%; iso-CIs :o, iso-c,, and anteiso-C, :o acids are the major cellular fatty acids. The type strain is strain IAM 13621. 0001-4688 0 1988 SGM

A thermostable tryptophanase was extracted from a thermophilic bacterium, Symbiobacterium thermophilum strain T, which is obligately symbiotic with the thermophilic Bacillus strain S. The enzyme was purified 21-fold to homogeneity with 19% recovery by a series of chromatographies using anion-exchange, hydroxylapatite, hydrophobic interaction, and MonoQ anion-exchange columns. The molecular weight of the purified enzyme was estimated to be approximately 210,000 by gel filtration, while the molecular weight of its subunit was 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits. The isoelectric point of the enzyme was 4.9. The tryptophanase was stable to heating at 65 degrees C for 20 min and the optimum temperature for the enzyme activity for 20 min reaction was 70 degrees C. The optimum pH was 7.0. The NH2-terminal amino acid sequence of this tryptophanase shows similarity to that of Escherichia coli K-12, despite a great difference in the thermostability of these two enzymes. The purified enzyme catalyzed the degradation (alpha, beta-elimination) of L-tryptophan into indole, pyruvate, and ammonia in the presence of pyridoxal-5'-phosphate. The Km value for L-tryptophan was 1.47 mM. 5-Hydroxy-L-tryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, and L-serine were also used as substrates and converted to pyruvate. The reverse reaction of alpha, beta-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammonium acetate.

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