p53 tumor suppressor is a transcription factor that functions, in part, through many of its downstream target genes. We have identified a p53-inducible gene by performing mRNA differential display on IW32 murine erythroleukemia cells containing a temperature-sensitive p53 mutant allele, tsp53 . Sequence analysis of the fulllength cDNA revealed its identity as the mouse homologue of the human thiamine transporter 1 (THTR-1). Induction of the mouse THTR-1 (mTHTR-1) mRNA was detectable as early as 1 h at 32.5°C; upon shifting back to 38.5°C, mTHTR-1 transcript was rapidly degraded with a half-life of less than 2 h. Elevation of mTHTR-1 expression was found in DNA damage-induced normal mouse embryonic fibroblast cells, but not in p53 ؊/؊ mouse embryonic fibroblast cells, suggesting that mTHTR-1 induction was p53-dependent. A region within the first intron of the mTHTR-1 gene bound to p53 and conferred the p53-mediated transactivation. Furthermore, increased thiamine transporter activities were found in cells overexpressing mTHTR-1 and under conditions of DNA damage or p53 activation. Our findings indicate that p53 may be involved in maintaining thiamine homeostasis through transactivation of THTR-1.

By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies of NtrC recognition sequence to N 18 )-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.Many bacteria are able to utilize acetoin as a carbon source. In some of the bacteria, dissimilation of acetoin proceeds via an oxidative cleavage process which is catalyzed by an acetoin dehydrogenase enzyme system with acetaldehyde and acetyl coenzyme A as the end products (13, 22). The acetoin dehydrogenase enzyme system is composed of E1␣ and E1␤, an acetoin-dependent dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR); E2, a dihydrolipoamide acetyltransferase; and E3, a dihydrolipoamide dehydrogenase. Structural genes encoding E1␣, E1␤, E2, and E3 of the acetoin dehydrogenase enzyme system have recently been isolated from several bacterial species (11,19,25,28). In Alcaligenes eutrophus, the structural genes identified for E1␣ (acoA), E1␤ (acoB), and E2 (acoC) are clustered with acoX, which encodes a protein of unknown function in the order of acoXABC (28). The structural gene for E3 which is not found in A. eutrophus aco operon, however, is present as acoL in Pelobacter carbinolicus acoABCLS (25) and Clostridium magnum acoABXCL (19) operons and as acoD in the Klebsiella pneumoniae acoABCD operon (27). Amino acid sequences deduced from these genes exhibit significant homology to those of the respective component of various 2-oxo acid dehydrogenase complexes, suggesting an evolutionary relationship among these catabolic systems.Despite the wealth of information on acetoin dehydrogenases, how these aco operons are regulated is less understood. The acoR gene located upst...

The acoD gene, which encodes a dihydrolipoamide dehydrogenase component of the acetoin dehydrogenase enzyme system of Klebsiella pneumoniae was isolated and the nucleotide sequence determined. The gene is capable of encoding a protein of 465 amino acid residues with conserved binding domains for NAD and FAD, and two redox-active cysteine residues. The acoD gene product exhibited a Michaelis constant of 170 microM for NAD, while NADP can not be used as a substrate. The purified enzyme appeared to be a dimer of the acoD gene product. It did not associate tightly with the E1 and E2 components of either acetoin dehydrogenase or 2-oxoglutarate dehydrogenase to form an active multi-enzyme complex.