Definition of the Bacillus subtilis PurR Operator Using Genetic and Bioinformatic Tools and Expansion of the PurR Regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO

doi:10.1128/JB.183.21.6175-6183.2001

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Journal of Bacteriology, November 2001, p. 6175-6183, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6175-6183.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Definition of the Bacillus subtilis PurR Operator Using Genetic and Bioinformatic Tools and Expansion of the PurR Regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO

Hans H. Saxild,¹^,^* Katja Brunstedt,¹ Karin I. Nielsen,¹ Hanne Jarmer,¹ and Per Nygaard²

BioCentrum-DTU, Technical University of Denmark, Lyngby,¹ and Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen,² Denmark

Received 12 June 2001/Accepted 13 August 2001

The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subjectto control by the purR gene product (PurR) and phosphoribosylpyrophosphate.This control is also exerted on the purA and purR genes. A consensussequence for the binding of PurR, named the PurBox, has been suggested(M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen,and D. Nilsson, J. Bacteriol. 180:3900-3906, 1998). To determinewhether the expression of other genes might be regulated by PurR,we performed a search for PurBox sequences in the B. subtilisgenome sequence and found several candidate PurBoxes. By the useof transcriptional lacZ fusions, five selected genes or operons(glyA, yumD, yebB, xpt-pbuX, and yqhZ-folD), all having a putativePurBox in their upstream regulatory regions, were found to beregulated by PurR. Using a machine-learning algorithm developedfor sequence pattern finding, we found that all of the genes identifiedas being PurR regulated have two PurBoxes in their upstream controlregions. The two boxes are divergently oriented, forming a palindromicsequence with the inverted repeats separated by 16 or 17 nucleotides.A computerized search revealed one additional PurR-regulated gene,ytiP. The significance of the tandem PurBox motifs was demonstratedin vivo by deletion analysis and site-directed mutagenesis ofthe two PurBox sequences located upstream of glyA. All six genesor operons encode enzymes or transporters playing a role in purinenucleotide metabolism. Functional analysis showed that yebB encodesthe previously characterized hypoxanthine-guanine permease PbuGand that ytiP encodes another guanine-hypoxanthine permease andis now named pbuO. yumD encodes a GMP reductase and is now namedguaC.

^* Corresponding author. Mailing address: BioCentrum-DTU, Section for Molecular Microbiology, Technical University of Denmark,Building 301, DK-2800 Lyngby, Denmark. Phone: 45 25 24 95. Fax:45 88 26 60. E-mail: hans.h.saxild{at}biocentrum.dtu.dk.

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