The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5 regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helixes, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.T hiamin pyrophosphate (TPP), also known as cocarboxylase, is the cofactor of key enzymes of carbon metabolism such as pyruvate dehydrogenase, ␣-ketoglutarate dehydrogenase, transketolase, pyruvate decarboxylase, and others. TPP is made by the enzymatic coupling of two independently synthesized precursors, 4-amino-5-hydroxymethyl-2-methyl pyrimidine pyrophosphate and 5-(2-hydroxyethyl)-4-methyl thiazole monophosphate (1).In Escherichia coli and Salmonella enterica serovar Typhimurium, several genes are known to participate in TPP biosynthesis (2). thiC and thiD are required for 4-amino-5-hydroxymethyl-2-methyl pyrimidine pyrophosphate synthesis (3, 4), whereas dxs (5, 6), thiF, thiS, thiG, thiH, and thiI are involved in 5-(2-hydroxyethyl)-4-methyl thiazole monophosphate synthesis (3,7,8). In addition, the product of the thiE gene couples 4-amino-5-hydroxymethyl-2-methyl pyrimidine pyrophosphate and 5-(2-hydroxyethyl)-4-methyl thiazole monophosphate to give thiamin monophosphate, which undergoes another phosphorylation catalyzed by the product of the thiL gene to form TPP, the biologically active form of vitamin B 1 (9, 10). A salvage enzyme (thiamin kinase) encoded by thiK, which incorporates exogenous thiamin into TPP (11), also exists. In both organisms the thi genes are located throughout the chromosome and are arranged in three operons and four single gene loci (2). thiCEFSGH, thiMD (ThiM and ThiD are involved in salvage of 5-(2-hydroxyethyl)-4-methyl thiazole and 4-amino-5-hydroxymethyl-2-methyl pyrimidine from the culture medium, respectively; ThiD also catalyzes the phosphorylation of 4-amino-5-hydroxymethyl-2-methyl pyrimidine monophosphate and thiBPQ (which code for an ABC type transport system for t...