ROLES OF THE 3 TRANSCRIPTIONAL ATTENUATORS OF THE BACILLUS-SUBTILIS PYRIMIDINE BIOSYNTHETIC OPERON IN THE REGULATION OF ITS EXPRESSION

Expression of the Bacillus subtilis pyr operon is regulated by exogeno us pyrimidines and the protein product of the first gene of the operon , PyrR. It has been proposed that PyrR mediates transcriptional attenu ation at three untranslated segments of the operon (R. J. Turner, Y. L u, and R. L. Switzer, J. Bacteriol., 176:3708-3722, 1994), In this stu dy, transcriptional fusions of the pyr promoter followed by the pyr at tenuation sequences, either individually or in tandem to a lacZ report er gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expres sion, These fusions were studied as chromosomal, integrants in various B. subtilis strains to examine the entire range of control by pyrimid ines, PyrR dependence, and developmental control of pyr gene expressio n. The nutritional regulation of each attenuator separately was roughl y equivalent to that of the other two and was totally dependent upon P yrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spae promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the co rresponding fusion containing the pyr promoter, demonstrating that att enuator-dependent regulation is independent of the promoter, Extreme p yrimidine starvation gave rise to two- to threefold-higher levels of e xpression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved, Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent reg ulation is not a specific property of the pyr operon. Conversion of th e initiator AUG codon in a small open reading frame in the pyrP:pyrB i ntercistronic region to UAG reduced expression by about half but did n ot alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism, Development al regulation of pyr expression during early stationary phase was foun d to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.