ANALYSIS AND EXPRESSION OF THE THRC GENE OF BREVIBACTERIUM-LACTOFERMENTUM AND CHARACTERIZATION OF THE ENCODED THREONINE SYNTHASE

The thrC gene of Brevibacterium lactofermentum was cloned by complemen tation of Escherichia coli thrC auxotrophs. The gene was located by de letion mapping and complementation analysis in a 2.9kb Sau3AI-HindIII fragment of the genome. This fragment also complemented a B. lactoferm entum UL1035 threonine auxotroph that was deficient in threonine synth ase. A 1,892-bp DNA fragment of this region was sequenced; this fragme nt contained a 1,446-bp open reading frame that encoded a 481-amino-ac id protein having a deduced M(r) of 52,807. The gene was expressed in E. coli, by using the phage T7 system, as a 53-kDa protein. The promot er region subcloned in promoter-probe plasmids was functional in E. co il. A Northern analysis revealed that the gene was expressed as a mono cistronic 1,400 nucleotide transcript. The transcription start point o f the thrC gene was located by S1 mapping 6 bp upstream from the trans lation initiation codon, which indicated that this promoter was one of the leaderless transcription-initiating sequences. The threonine synt hase overexpressed in B. lactofermentum UL1035 was purified almost to homogeneity. The active form corresponded to a monomeric 52.8 kDa prot ein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophor esis. The purified enzyme required pyridoxal phosphate as its only cof actor to convert homoserine phosphate into threonine.