Characterization of cis-acting mutations in the first attenuator region of the Bacillus subtilis pyr operon that are defective in pyrimidine-mediated regulation of expression

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Abstract
A transcriptional attenuation mechanism for the regulation of pyr operon expression in Bacillus subtilis in which the PyrR regulatory protein binds pyr mRNA at three sites with similar sequences to cause transcription termination in response to elevated pyrimidine nucleotide pools has been proposed (R. J. Turner, Y. Lu, and R. L. Switzer, J. Bacteriol. 176:3708-3722, 1994). Twenty-seven mutants with cis-acting defects in the repression by pyrimidines of beta-galactosidase expression of a pyr-lacZ fusion-integrant were isolated as blue colonies on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) agar plates containing uracil and uridine after UV irradiation or treatment with mutagens or following mutD mutagenesis. These mutants showed normal repression of the chromosomal pyr operon by exogenous pyrimidines. Sequence analysis revealed 12 unique sites of mutation, which occurred in the conserved putative PyrR binding sequence (10 of the 12) or in the stem of the transcriptional terminator structure. These mutants strongly support the proposed model for regulation of the pyr operon.