Analysis of the methionine biosynthetic pathway in the extremely thermophilic Eubacterium Thermus thermophilus

Four DNA fragments that could rescue the mutations of four Met(-) mutants w ere cloned from Thermus thermophilus HB27 and their complete nucleotide seq uences were determined. Two of the four fragments respectively contained th e greater parts of the metF and metH genes, the predicted amino acid sequen ces of which showed identities of 30.8% and 32.7% with 5,10-methylenetetrah ydrofolate reductase (EC 1.7.99.5) and vitamin B-12-dependent homocysteine transmethylase (EC 2.1.1.13) of Escherichia coil. The other two DNA fragmen ts, which overlapped one another, contained two open reading frames whose p redicted amino acid sequences were respectively similar to those of O-acety lhomoserine sulfhydrylase (EC 4.2.99.10, the product of the MET17 gene) and homoserine O-acetyltransferase (EC 2.3.1.31, the product of the MET2 gene) of Saccharomyces cerevisiae. The metF, metH, MET2, and MET17 genes of T. t hermophilus were disrupted by introducing the heat-stable kanamycin nucleot idyltransferase gene into the genome. Each transformant showed methionine a uxotrophy. Both the MET2- and MET17-disrupted mutants could grow in a minim al medium containing homocysteine but not in the same medium containing suc cinylhomoserine or cystathionine. In contrast, the metF- and metH-disrupted mutants could not grow in the minimal medium containing homocysteine. Thes e results suggest that in T. thermophilus, homoserine is directly converted to homocysteine via O-acetylhomoserine and that homocysteine is methylated to synthesize methionine.