TRANSCRIPTIONAL ANALYSIS AND REGULATORY SIGNALS OF THE HOM-THRB CLUSTER OF BREVIBACTERIUM-LACTOFERMENTUM

Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustere d in the chromosome of Brevibacterium lactofermentum in the order 5' h om-thrB 3', separated by only 10 bp. The Brevibacterium thrB gene is e xpressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium glutamicum and complements auxotrophs of all three or ganisms deficient in homoserine kinase, whereas the Brevibacterium hem gene did not complement two different E. cell auxotrophs lacking homo serine dehydrogenase. However, complementation was obtained when the h omoserine dehydrogenase was expressed as a fusion protein in E. coli. Northern (RNA) analysis showed that the hom-thrB cluster is transcribe d, giving two different transcripts of 2.5 and 1.1 kb. The 2.5-kb tran script corresponds to the entire cluster hom-thrB (i.e., they form a b icistronic operon), and the short transcript (1.1 kb) originates from the thrB gene. The promoter in front of horn and the hom-internal prom oter in front of thrB were subcloned in promoter-probe vectors of E. c oli and corynebacteria. The thrB promoter is efficiently recognized bo th in E. coli and corynebacteria, whereas the horn promoter is functio nal in corynebacteria but not in E. coli. The transcription start poin ts of both promoters have been identified by primer extension and S1 m apping analysis. The thrB promoter was located in an 87-bp fragment th at overlaps with the end of the hem gene. A functional transcriptional terminator located downstream from the cluster was subcloned in termi nator-probe vectors.