Regulation and Expression of the Pyrimidine Operon of the Extreme Thermophilic Bacterium Thermus Z05
Pingguo CHEN*a, Mark VAN DE CASTEELEa, and Nicolas GLANSDORFFa,b
a Microbiology, Free University of Brussels (VUB) and Flanders Interuniversity Institute for Biotechnology, Ave E. Gryson 1, B-1070 Brussels, Belgium
b Research Institute of the CERIA, Ave E. Gryson 1, B-1070 Brussels, Belgium
The pyrimidine operon of Thermus Z05 contains four genes in the order pyrR, pyrB, bbc (an unidentified open reading frame), and pyrC (1). The transcription initiation site was mapped at about 115 nucleotides upstream of the pyrR translation start codon. The cognate Thermus pyr promoter also functions in heterologous expression of Thermus pyr genes in E. coli. The pyr promoter sequence corresponds closely to the consensus promoters described in Thermus and E. coli. In Thermus Z05, pyrB and pyrC gene expression is repressed by uracil, and pyrC gene expression is derepressed more than 10 fold in a Thermus Z05 pyrB mutant.
Previous studies suggested that Thermus pyr genes were regulated by attenuation (2). Regulation would occur via the occlusion of a sequence overlapping the ribosome-binding site of a leader preceding a transcription terminator. Thus, in contrast to pyr attenuation in two Bacillus spp. (3,4), control of the Thermus pyr gene cluster would not involve an antiterminator structure. Strong amino acid sequence identities (about 60%) between Thermus pyrR and the pyrR attenuation protein of Bacillus suggested however that the same type of regulatory protein was involved in Thermus and in Bacillus. We investigated this regulation by fusing the pyr promoter and the surrounding sequences to a promoterless chloramphenicol acetyltransferase gene (CAT) in E. coli. The minimal promoter region that could be isolated on one fragment included the sequence from-68 to +7. The regulation of the pyr operon in response to excess pyrimidine or orotic acid (a limiting pyrimidine source) was examined in the presence of the pyrR protein expressed in E. coli. Our results suggest that binding of the pyrR protein to the leader RNA regulates transcription termination over about a 7-fold range. Furthermore, disruption of the pyrR gene abolished this regulation. To verify that the altered regulatory regions behave as a pyrR protein binding site, studies on mutations engineered in the leader region of the Thermus pyr operon are in progress.
Comparison of the sequence from E. coli upp and Bacillus spp. pyrR, as well as direct enzymatic assay, demonstrated that Thermus Z05 pyrR also has uracil phosphoribosyltransferase activity. A 22 kDa pyrR protein could be expressed in E. coli from the T7 phage promoter under the control of the T7 RNA polymerase.
1. Van de Casteele, M., Desmarez, L., Legrain, C., Chen, P.G., Van Lierde, K., Pierard, A., and Glansdorff, N. (1994) Biocatalysis, 11, 165-179.
2. Van de Casteele, M., Chen, P.G., Roovers, M., Legrain, C., and Glansdorff, N. (1997). J. Bacteriol., 179, 3470-3481.
3. Turner, R.J., Lu, Y., and Switzer, R.L. (1994) J. Bacteriol.,176, 3708-3722.
4. Ghim, S.Y., Nielsen, P., and Neuhard, J. (1994) Microbiology, 140, 479-491.
