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; Katja Brunstedt; Karin I Nielsen; Hanne Jarmer; Per Nygaard

doi:10.1128/JB.183.21.6175-6183.2001

. 2001 Nov;183(21):6175–6183. doi: 10.1128/JB.183.21.6175-6183.2001

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, Per Nygaard ²

PMCID: PMC100094 PMID: 11591660

Abstract

The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subject to control by the purR gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the purA and purR genes. A consensus sequence 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 determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the B. subtilis genome sequence and found several candidate PurBoxes. By the use of transcriptional lacZ fusions, five selected genes or operons (glyA, yumD, yebB, xpt-pbuX, and yqhZ-folD), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR. Using a machine-learning algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two PurBoxes in their upstream control regions. The two boxes are divergently oriented, forming a palindromic sequence 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 demonstrated in vivo by deletion analysis and site-directed mutagenesis of the two PurBox sequences located upstream of glyA. All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism. Functional analysis showed that yebB encodes the previously characterized hypoxanthine-guanine permease PbuG and that ytiP encodes another guanine-hypoxanthine permease and is now named pbuO. yumD encodes a GMP reductase and is now named guaC.

We have detailed knowledge about the regulation of expression of the pur genes, which encode enzymes of the purine biosynthetic pathway in bacteria (24, 25). In Escherichia coli, the pur genes are scattered on the chromosome and are found as single genes or small operons. A regulatory protein, the PurR repressor, of the LacI type of regulatory proteins, regulates the expression of the pur genes or operons. When E. coli grows in the presence of guanine or hypoxanthine, these compounds are taken up and salvaged and at the same time they bind to the PurR repressor. PurR binds to a 16-bp palindromic sequence that overlaps the −35 promoter region of the pur genes (11) and inhibits transcription of the pur genes.

In B. subtilis, the genes encoding the biosynthesis of IMP are located in the pur operon. Three other genes (purA, guaA, and guaB) required for AMP and GMP synthesis are located as single genes. Expression of the pur operon is subject to dual regulation of transcription termination and transcription initiation. Termination of transcription is regulated by a termination-antitermination mechanism in a 242-nucleotide mRNA leader region preceding the first gene of the pur operon (5). The termination mechanism is triggered by guanine or hypoxanthine; however, the molecular mechanism has not been clarified. Initiation of transcription of the pur operon, and also of the purA and purR genes, is repressed in response to the presence of adenine in the culture medium (5). Addition of adenine results in lowering of the cellular pool of the low-molecular-weight effector molecule phosphoribosylpyrophosphate (PRPP) (15). Two regulatory elements are required for this regulation, the PurR repressor and a DNA operator site for repressor binding. PurR binding to the operator site is blocked by PRPP. The PurR protein is a 62-kDa homodimer (17, 21) that—as judged by footprinting analysis—interacts with a region between −149 and −29 relative to the transcriptional start site of the pur operon (17). A second protein encoded by yabJ, which is located in an operon with purR, has been suggested to act together with the PurR repressor (10).

Recently, a regulatory protein, also named PurR, that activates pur gene transcription was identified in Lactococcus lactis. L. lactis PurR shows extensive amino acid sequence identity with the B. subtilis PurR repressor (7). The L. lactis PurR protein binds upstream of the promoter region of pur genes (6). Based on genetic analysis and sequence comparison between the nucleotide sequences upstream of genes in B. subtilis and L. lactis, Kilstrup and coworkers were able to suggest a possible cis-acting sequence (5′-AWWWCCGAACWWTH-3′), named the PurBox, which is required for PurR-mediated control of gene expression (6).

In the present work, we provide evidence that an operator site comprised of two PurBoxes is required for PurR control in B. subtilis and that other genes of importance for purine synthesis are also regulated by PurR.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media.

The bacterial strains and plasmids used in this work are listed in Table 1. DNA primers used for PCR amplification and primer extension are listed in Table 2. B. subtilis was grown in Spizizen minimal salt medium supplemented with 0.2% l-glutamate, 40 mg of l-tryptophan per liter, and 1 mg of thiamine per liter and with 0.4% glucose as a carbon source. Purine compounds (adenine and guanosine) were added to a final concentration of 1 mM. For selection of antibiotic resistance, antibiotics were used at the following concentrations: ampicillin, 100 mg/liter; neomycin, 5 mg/liter; erythromycin, 1 mg/liter; lincomycin, 25 mg/liter; chloramphenicol, 6 mg/liter.

TABLE 1.

Strains and plasmids used in this work

Strain or plasmid	Genotype or characteristics	Source or reference
B. subtilis
168	trpC2	C. Anagnostopoulos^a
168/pMAP65	trpC2/pMAP65	168 transformed with pMAP65 selecting for Neo^r
HH355	trpC2 yumD::[pTM007 yumD′-lacZ (erm)]	168 transformed with pTM007 selecting for Er^r
HH413	trpC2 glyA::[pHH1101 (cat)]	168 transformed with pHH1101 selecting for Cm^r
HH417	trpC2 amyE::[pHH1108 pbuG′-lacZ (cat)]	168 transformed with pHH1108 selecting for Cm^r
HH418	trpC2 amyE::[pHH1109 xpt′-lacZ (cat)]	168 transformed with pHH1109 selecting for Cm^r
HH419	trpC2 amyE::[pHH1108 pbuG′-lacZ (cat)] purR::neo	HH417 transformed with LCC28 selecting for Neo^r
HH420	trpC2 amyE::[pHH1109 xpt′-lacZ (cat)] puR::neo	HH418 transformed with LCC28 selecting for Neo^r
ED448	trpC2 yqhZ::pMutin4 pMAP65	168/pMAP65 transformed with YQHZd selecting for Er^r
ED449	trpC2 ywoE::pMutin1 pMAP65	168/pMAP65 transformed with BFA2232 selecting for Er^r
ED453	trpC2 ytiP::pMutin1 purR::neo	BFA2025 transformed with LCC28 selecting for Neo^r
KB-3A	trpC2 amyE::[pKB3-1 purE′-lacZ (cat)]	168 transformed with pKB3-1 selecting for Cm^r
KB-3Am	trpC2 amyE::[pKB3-1 purE′-lacZ (cat)] purR::neo	KB-3A transformed with LCC28 selecting for Neo^r
KB-4C	trpC2 amyE::[pKB4-4 rapB′-lacZ (cat)]	168 transformed with pKB4-4 selecting for Cm^r
KB-4Cm	trpC2 amyE::[pKB4-4 rapB′-lacZ (cat)] purR::neo	KB-4C transformed with LCC28 selecting for Neo^r
KB-5D	trpC2 amyE::[pKB5-4 yqhZ′-lacZ (cat)]	168 transformed with pKB5-4 selecting for Cm^r
KB-5Dm	trpC2 amyE::[pKB5-4 yqhZ′-lacZ (cat)] purR::neo	KB-5D transformed with LCC28 selecting for Neo^r
PEH03	trpC2 amyE::[pPEH04 glyA′-lacZ (cat)] purR::neo	PEH06 transformed with LCC28 selecting for Neo^r
PEH06	trpC2 amyE::[pPEH04 glyA′-lacZ (cat)]	168 transformed with pPEH04 selecting for Cm^r
PEH07	trpC2 amyE::[pPEH06 glyA′ (G₋₁₁₀→C)-lacZ (cat)]	168 transformed with pPEH06 selecting for Cm^r
KN05n	trpC2 amyE::[pKN05n glyA′-lacZ (neo)]	168 transformed with pKN05n selecting for Neo^r
PEH08	trpC2 amyE::[pPEH05 glyA′ Δ(A₋₁₂₀–T₋₉₉)-lacZ (cat)]	168 transformed with pPEH05 selecting for Cm^r
KN07c	trpC2 amyE::[pKN07c glyA′ (C₋₇₈→G)-lacZ (cat)]	168 transformed with pKN07c selecting for Cm^r
KN08c	trpC2 amyE::[pKN08c glyA′ (T₋₇₆→G)-lacZ (cat)]	168 transformed with pKN08c selecting for Cm^r
KN09c	trpC2 yabJ::[pKN09-2 (cat)]	168 transformed with pKN09-2 selecting for Cm^r
KN015cn	trpC2 amyE::[pKN05n glyA′-lacZ (neo)] yabJ::[pKN09-2 (cat)]	KN05c transformed with pKN09-2 selecting for Cm^r
LCC28	purR::neo	2
TM307	trpC2 yumD::[pTM007 yumD′-lacZ (erm)] purR::neo	HH355 transformed with LCC28 selecting for Neo^r
YQHZd	trpC2 yqhZ::pMutin4	K. Kobayashi^b
BFA2025	trpC2 ytiP::pMutin1	Micado database^c
BFA2026	trpC2 ytjP::pMutin1	Micado database
BFA2232	trpC2 ywoE::pMutin1	Micado database
BFA2255	trpC2 yebB::pMutin1	Micado database
E. coli MC1061	F⁻araD139 Δ(ara-leu)7696 galE15 galK16 Δ(lac)X74 rpsL (Str^r) hsdR2 (r⁻ m⁻) mcrA mcrB	Laboratory stock
Plasmids
pMutin4	Ap^r (E. coli) or Er^r (B. subtilis); integrational vector for knockout mutations and formation of transcriptional lacZ fusions; IPTG-inducible P_spac promoter introduced to ensure expression of downstream genes	20
pMAP65	Neo^r (B. subtilis); plasmid overexpressing LacI	9
pDG268neo	Ap^r (E. coli) or Neo^r (B. subtilis); vector used for integration of transcriptional lacZ fusions into amyE gene of B. subtilis	12
pDG268cat	Ap^r (E. coli) or Cm^r (B. subtilis); vector used for integration of transcriptional lacZ fusions into amyE gene of B. subtilis	12
pBOE335	Ap^r (E. coli) or Cm^r (B. subtilis); integrational vector, pUC19 containing cat gene cloned into KasI site	12
pTM007	pMutin4 digested with HindIII and BamHI and ligated to a PCR fragment (primers 18 and 19) digested with the same enzymes	This work
pHH1101	pBOE335 digested with EcoRI and BamHI and ligated to a glyA internal PCR fragment (primers 8 and 9) digested with the same enzymes	This work
pHH1108	pDG268cat digested with HindIII and BamHI and ligated to a PCR fragment (primers 14 and 15) digested with the same enzymes	This work
pHH1109	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 12 and 13) digested with the same enzymes	This work
pKB3-1	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 16 and 17) digested with the same enzymes	This work
pKB4-4	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 10 and 11) digested with the same enzymes	This work
pKB5-4	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 20 and 21) digested with the same enzymes	This work
pPEH04	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 1 and 6) digested with the same enzymes	This work
pPEH06	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 2 and 6) digested with the same enzymes	This work
pPEH05	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 3 and 6) digested with the same enzymes	This work
pKN07c	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 5 and 6) digested with the same enzymes	This work
pKN08c	pDG268cat digested with EcoRI and BamHI and ligated to a PCR fragment (primers 4 and 6) digested with the same enzymes	This work
pKN05n	pDG268neo digested with EcoRI and BamHI and ligated to a PCR fragment (primers 1 and 6) digested with the same enzymes	This work
pKN09-2	pBOE335 digested with EcoRI and ligated to a yabJ internal PCR fragment (primers 22 and 23) digested with the same enzyme	This work

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Centre National de la Recherche Scientifique, Jouy-en-Josas, France.

http://bacillus.genome.ad.jp/.

http://locus.jouy.inra.fr/cgi-bin/genmic/madbase_home.pl.

TABLE 2.

DNA primers used in this work

Gene and primer no.	5′-linked restriction site sequence	Nucleotide sequence^a	Coordinates^b
glyA
1	EcoRI	5′-GCCGGAATTCAATAAATTCCGAACTTTAAATTA-3′	(−120)–(−98)
2	EcoRI	5′-GCCGGAATTCAATAAATTCCCAACTTTAAATTA-3′	(−120)–(−98)
3	EcoRI	5′-GCCGGAATTCAAATTTAAGTTATTAATATTCG-3′	(−98)–(−77)
4	EcoRI	5′-GCCGGAATTCAATAAATTCCGAACTTTAAATTAAATTTAAGTTA TTAATATTCGGTTTTA-3′	(−120)–(−72)
5	EcoRI	5′-GCCGGAATTCAATAAATTCCGAACTTTAAATTAAATTTAAGTTA TTAATATTGGTTTTTA-3′	(−120)–(−72)
6	BamHI	5′-GCGGGATCCTTAGTCTGTTGGCGTTC-3′	(+118)–(+102)
7	None	5′-TTAGTCTGTTGGCGTTC-3′	(+118)–(+102)
8	EcoRI	5′-GCCGGAATTCTTTGCTTGTGCGCC-3′	3789200–3789213
9	BamHI	5′-GCGGGATCCCAAGTGTTTAACGCC-3′	3789465–3789451
rapB
10	EcoRI	5′-GCCGGAATTCATACGTAGAAAAACCG-3′	3771315–3771300
11	BamHI	5′-GCGGGATCCGGCCATGCCCCTCC-3′	3771183–3771196
xpt
12	EcoRI	5′-GCCGGAATTCCGAATCCCCTTGAAATACG-3′	2319557–2319539
13	BamHI	5′-GCGGGATCCCGCGATTATATGAGTG-3′	2319423–2319438
pbuG
14	HindIII	5′-GCCGAAGCTTGTTTATTACGAACAAAATCCG-3′	693858–693878
15	BamHI	5′-GCGGGATCCTTCCCGCGATTATACG-3′	693980–693956
purE
16	EcoRI	5′-GCCGGAATTCAGATCGTTCCGTGCGGG-3′	697749–697765
17	BamHI	5′-GCGGGATCCGATTATATGAGGTCGTG-3′	697907–693956
yumD
18	HindIII	5′-GCCGAAGCTTTCCGTACGTTTACCGCC-3′	3302180–3302196
19	BamHI	5′-GCGGGATCCACAGCATTTGAGTGCCC-3′	3302478–3302462
yqhZ-folD
20	EcoRI	5′-GCCGGAATTCTTTCTTCATGAACGTG-3′	2529102–2529087
21	BamHI	5′-GCGGGATCCCATTTTCTTTCTCCTTTG-3′	2528935–2529952
yabJ
22	EcoRI	5′-GCCGGAATTCGGCCAAATCCCTTTGACTCC-3′	55413–55432
23	EcoRI	5′-GCCGGAATTCAACTGTTCCATATCCGCG-3′	55544–55527

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Each underlined sequence indicates the position of the 5′-linked restriction site. Letters in boldface (in primers 2, 4, and 5) indicate mutational changes.

Numbers in parentheses indicate nucleotide positions relative to the +1 transcriptional start site of glyA. Other numbers indicate the genome sequence coordinates (8) of the primer 5′ and 3′ ends, respectively.

Nucleic acid manipulation and genetic techniques.

Isolation of DNA and RNA and basic molecular biology techniques were performed as previously described (12, 13, 26).

Construction of transcriptional lacZ fusions.

Different promoter-containing PCR products were generated by using the primer combinations listed in Tables 1 and 2. The various DNA fragments were digested with restriction enzymes and ligated into pDG268cat or pDG268neo digested with the same enzymes and transformed into E. coli MC1061 selecting for Ap^r. Plasmids extracted from E. coli were integrated into the B. subtilis chromosome as described before (12). The yumD′-lacZ fusion was constructed by amplifying an internal segment of the yumD gene and cloning it in front of the lacZ gene of pMutin4 (20). The resulting plasmid was transformed into the B. subtilis yumD locus by selecting for Er^r as described by Vagner and coworkers (20).

Enzyme assays and measurement of purine base uptake.

Cell extracts were made as described before (15). Serine hydroxymethyltransferase (SHMT) activity was determined in a coupled assay using l-allo-threonine as the substrate (16). GMP reductase activity was determined by measuring the formation of [¹⁴C]IMP from [¹⁴C]GMP. A 50-μl volume of assay buffer contained 0.2 mM NADPH, 0.1 mM [¹⁴C]GMP (50 mCi/mmol), and glucose-6-phosphate dehydrogenase (10 U), as well as 2 mM glucose-6-phosphate to regenerate NADPH. Cell extract was added, and after 1, 2, 4, and 8 min, 5-μl samples were removed and spotted on a polyethyleneimine-impregnated thin-layer chromatography plate (Merck, Darmstadt, Germany). The chromatogram was dried and developed in 0.4 M phosphate buffer (pH 3.4) to separate IMP from GMP. The plate was dried, and radioactivity was measured in an InstantImager (Packard). β-Galactosidase activity was determined as described previously (2). All enzyme determinations were repeated at least three times. Enzyme activity is given as nanomoles of product formed per minute (equals 1 U). Total protein was determined by the method of Lowry et al. Uptake of purine bases was performed as described by Saxild and Nygaard (14).

Bioinformatic tools.

Searches for specific nucleotide sequences in the B. subtilis genome were performed by using the WinSeq computer software developed by Flemming Hansen (unpublished). The machine-learning algorithm ANN-Spec, which was designed to discover ungapped patterns in DNA sequences (23), was used to analyze the B. subtilis genome sequence for the presence of the tandem-PurBox sequence. The computer program for UNIX systems is available from us.

RESULTS

Initial search for potential PurR binding sites on the B. subtilis chromosome.

The PurR binding sequence (PurBox) 5′-AWWWCCGAACWWTH-3′ (6) was used as the query sequence in a computerized search of the B. subtilis genome using the WinSeq computer software. An alignment of the B. subtilis PurBox sequences upstream of the pur operon, purA and purR, revealed that the purR and pur operon PurBoxes diverge from the consensus sequence at one and two positions, respectively. These three positions were therefore considered less important and were assigned a low-importance weight, whereas all other positions were assigned a high-importance weight. Using these search parameters, we found 249 potential PurBoxes with zero, one, or two low-weight mismatches or one high-weight mismatch. Because PurR is reported to bind to a regulatory region upstream of the affected genes, the locations of the 249 potential PurBoxes were examined. Those PurBoxes that are located 0 to 350 nucleotides upstream of the start codon of the downstream open reading frame (ORF) were selected. This assortment resulted in 46 ORFs.

Test for possible PurR control of the expression of six selected operons.

Among the 46 ORFs, six operons or genes, including xpt-pbuX, yebB, glyA, yumD, yqhZ-folD, and rapB, were selected for further analysis. The upstream control regions containing the putative PurBox sequence were cloned in front of lacZ in plasmid pDG268(cat) or pMutin4 and inserted into the chromosome. A fusion of the pur operon promoter was also constructed and used as a positive control (amyE::purE′-lacZ). An isogenic series of strains was constructed that contains the respective lacZ fusions in a purR genetic background. All strains were grown in minimal medium with or without adenine, and the β-galactosidase level was determined. The expression of all of the genes, except rapB, was repressed two- to threefold in the presence of adenine, and the levels were increased in a purR genetic background (Table 3). When the DNA sequences of the upstream regulatory regions of the identified PurR-controlled genes were compared, it became evident that they all contain two divergently oriented PurBox-like sequences separated by 16 or 17 nucleotides (Fig. 1). From the alignment of putative PurBoxes upstream of the six selected operons or genes, it appears that of all the PurR-regulated genes are preceded by one PurBox sequence with relatively high sequence similarity to the consensus sequence and by another PurBox sequence that has a more degenerated sequence. The upstream region of rapB contains only one PurBox sequence.

TABLE 3.

Effect of purine repressor PurR on expression of selected B. subtilis genes having putative PurBox sequences in their regulatory regions

Strain	Relevant genotype	β-Galatosidase activity (U/mg of protein)^a
Strain	Relevant genotype	MM^b	MM + Adenine
KB-3A	amyE::purE′-lacZ	145	49
KB-3Am	amyE::purE′-lacZ purR	341	228
HH418	amyE::xpt′-lacZ	92	56
HH420	amyE::xpt′-lacZ purR	338	292
HH417	amyE::yebB′-lacZ	164	62
HH419	amyE::yebB′-lacZ purR	635	507
PEH06	amyE::glyA′-lacZ	402	158
PEH03	amyE::glyA′-lacZ purR	1,815	1,790
HH355	yumD::[pTM007 yumD′-lacZ]	23	7
TM307	yumD::[pTM007 yumD′-lacZ] purR	422	391
KB-5D	amyE::yqhZ′-lacZ	251	113
KB-5Dm	amyE::yqhZ′-lacZ purR	682	670
KB-4C	amyE::rapB′-lacZ	15	10
KB-4Cm	amyE::rapB′-lacZ purR	7	9

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Values are means of three experiments. The variation was less than 20%.

MM, glucose minimal medium.

FIG. 1 — Alignment of the tandem-PurBox motif located upstream of nine PurR-regulated genes or operons. Only one DNA strand is shown. Boxed sequences are individual PurBox sequences. Shaded positions indicate nucleotides (nt) that diverge from the 5′-AWWWCCGAACWWTH-3′ consensus sequence defined by Kilstrup and coworkers (6). Letters in the two bottom boxes show nucleotides that are conserved in the tandem PurBox motif. Lightface letters indicate nucleotides that are conserved in eight of the nine PurBoxes, and boldface letters indicate nucleotides that are conserved in all nine PurBoxes.

Computerized search for regulatory regions containing the tandem PurBox motif.

We then wanted to test whether a refined computer search using the novel information about the PurR binding motif could identify the expected PurR-regulated genes and perhaps predict new genes that did not appear in the initial search. Potential promoter-containing DNA sequences in the B. subtilis genome were organized in a list of 4,222 entries, each containing a 400-nucleotide sequence upstream of one of the 4,222 predicted ORFs in the B. subtilis genome. This list was searched for sequences having the tandem PurBox motif. Using the ANN-Spec bioinformatics software (23), a weight matrix for the PurBox sequence was calculated on the basis of the sequence of a total of 16 PurBoxes located pairwise upstream of purR, purA, yqhZ-folD, yumD, purE, glyA, xpt-pbuX, and yebB, respectively. The program calculates an arbitrary score for each of the potential PurBox sequences. The file containing the 4,222 potential promoter regions was searched for sequences having two potential PurBoxes separated by no less than 11 and no more than 21 nucleotides. A total of 129 sequences were found, and Table 4 lists the 10 top-ranked loci for which the upstream 400-bp sequence contains the tandem PurBox motif having one or two PurBox sequences with a high score. For the remaining 116 loci, the scores for one or both potential PurBoxes were below the level of significance. As expected, the program identified all of the genes that were used to calculate the weight matrix. The upstream region of ytiP and ytjP was also found to contain a potential tandem PurBox motif with the correct spacing of 16 or 17 nucleotides between the PurBox sequences. ytiP and ytjP are divergently oriented on the chromosome and are separated by a 96-bp intercistronic region. The two potential PurBoxes are located closest to the ytiP reading frame. The 432-amino-acid primary sequence of YtiP is 47% identical to yebB of B. subtilis. The 463-amino-acid primary sequence of YtjP is 40% identical to a dipeptidase from L. lactis (472 amino acids, accession no. AAC45369). In order to analyze whether the genes are subject to purine control, BFA2025 (ytiP) and BFA2026 (ytjP) were grown in minimal medium supplemented with adenine or guanosine. The basal level in BFA2025 was 22 U/mg of protein, and the expression was repressed by adenine (to 9 U/mg of protein) and induced by guanosine (to 49 U/mg of protein). ytjP (BFA2026) expression did not respond to addition of purines (data not shown). Inactivation of purR in BFA2025 (strain ED453) resulted in derepression of ytiP expression both in the presence of adenine (66 U/mg of protein) and in the absence of adenine (81 U/mg of protein). We therefore concluded that ytiP, but not ytjP, belongs to the PurR regulon.

TABLE 4.

The 10 genes and reading frames (out of 129 candidates) in the B. subtilis genome showing the highest scores for the upstream twin PurBox sequences identified by the ANN-Spec bioinformatic software^a

Locus	PurBox score		Distance (nt)
Locus	Gene distal	Gene proximal	Distance (nt)
purA	6.44	12.78	17
glyA	10.23	12.22	17
purR	9.95	11.92	16
yqhZ	10.49	11.81	16
yumD	9.31	11.75	16
yebB	9.21	11.35	16
xpt	11.30	10.01	16
ytiP	8.95	9.57	17
purE	10.98	8.38	16
ytjP	10.32	7.89	17

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The ANN-Spec bioinformatic software was described by Workman and Stormo (23).

nt, nucleotides.

cis-acting elements involved in PurR repression of glyA expression.

The cis-acting requirements for PurR control of glyA expression was studied in more detail. The glyA transcriptional start site was determined in a primer extension experiment (data not shown), and the site is indicated in Fig. 2. Putative ς^A −10 and −35 regions are located at suitable distances upstream of the +1 position. The DNA fragment covering the region from −120 to +118 (Fig. 2) directed PurR-regulated transcription when fused to lacZ in a wild-type genetic background (PEH06, Table 3). The same fusion was constitutively expressed in a purR genetic background (PEH03, Table 3). A fusion with a DNA fragment with nucleotides −120 to −99 deleted was also constitutively expressed (PEH08, Table 5), indicating that the deduced PurBox (nucleotides −116 to −103 in Fig. 2) is required for PurR control. A G₊₁₁₀→C substitution was introduced into the −120 to +118 fragment, and this also leads to constitutive expression. This observation demonstrates the essential role of the central CG pair of the promoter-distal PurBox in mediating the negative control of gene expression by PurR. The promoter-proximal PurBox sequence was altered in two ways. T₊₇₆ was replaced with a G, and in theory, this should create a more consensus-like PurBox sequence. C₊₇₈ was replaced with a G, and in theory, this should result in a less consensus-like PurBox sequence. When fused to lacZ, the fragment containing the T₊₇₆→G mutation mediated a stronger repression by PurR in medium with adenine present whereas a fusion with the fragment containing the C₊₇₈→G mutation reduced repression by adenine to 1.3-fold, compared to 2.7-fold repression in the wild type.

FIG. 2 — Organization of the *glyA* regulatory region. Italic boldface letters indicate nucleotides constituting the tandem-PurBox motif. Boldface roman letters indicate the translational start codon of the *glyA* reading frame. Arrows and letters above the PurBox sequences indicate base pair substitutions in the various strains described in Table 5. The Δ symbol surrounded by dashed lines indicates the extent of the PurBox deletion in strain PEH08 (Table 5). Lines above the sequence indicate the locations of the putative −10 and −35 regions of the *glyA* promoter. The designation +1 indicates the transcriptional start site determined by primer extension analysis of *glyA* mRNA from cells grown in glucose minimal medium using primer 7 (Table 2).

TABLE 5.

Effects of mutational changes in the glyA regulatory region on the expression of a glyA-lacZ fusion^a

Strain	Relevant genotype	Change in glyA regulatory region	Adenine added	β-Galactosidase activity (U/mg of protein)
PEH06^b	amyE::glyA′-lacZ	None	−	402 ± 47
			+	158 ± 8
PEH08	amyE::glyA′ Δ(A₋₁₂₀–T₋₉₉)-lacZ	A₋₁₂₀ to T₋₉₉ deletion	−	2,176 ± 66
			+	2,182 ± 79
PEH07	amyE::glyA (G₋₁₁₀→C)-lacZ	G₋₁₁₀→C substitution	−	2,017 ± 123
			+	1,803 ± 93
KN08c	amyE::glyA (T₋₇₆→G)-lacZ	T₋₇₆→G substitution	−	428 ± 60
			+	82 ± 10
KN07c	amyE::glyA (C₋₇₈→G)-lacZ	C₋₇₈→G substitution	−	2,310 ± 346
			+	1,550 ± 387

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Strains were grown in glucose minimal medium plus and minus adenine (1 mM).

Values are from Table 3.

Is the yabJ gene product involved in the regulation of expression of PurR-controlled genes?

The yabJ gene located downstream of the purR gene has been suggested to encode a protein involved in the adenine-mediated repression of purA gene expression (10), although this was not observed when the purR-yabJ operon was first identified (21). To investigate whether the expression of the glyA gene was altered in a yabJ mutant, we determined the effects of adenine and guanosine on glyA expression in both the wild type and a yabJ mutant strain. As a control, we determined purA gene expression (Table 6). However, we found that adenine repression and guanosine induction of both genes were similar in wild-type strains and yabJ mutant strains. This finding favors the view that the yabJ gene product has no effect on glyA and purA gene expression.

TABLE 6.

Effect of yabJ disruption on expression of glyA and purA^a

Relevant genotype	Enzyme activity (U/mg of protein)^b
	MM		MM + adenine		MM + guanosine
	glyA	purA	glyA	purA	glyA	purA
Wild type	576	2.1	226	0.7	1,272	4.2
yabJ	572	2.5	265	1.0	1,121	5.9

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Cells were grown in minimal medium (MM) with purines added at 1 mM.

glyA expression was determined in strains KNO5n and KN015cn (yabJ), both containing a glyA-lacZ transcriptional fusion in the amyE gene; purA expression was determined in strains 168 and KNO9c (yabJ) by measuring adenylosuccinate synthetase activity. Values are means of three experiments. The variation was less than 20%.

Function of PurR-controlled genes glyA, yumD, yqhZ-folD, yebB, and ytiP

The derived amino acid sequence of glyA has high amino acid sequence similarity to SHMT from E. coli (accession no. P00477). In agreement with this, glyA mutant strain HH413 required glycine for growth. The SHMT levels were determined in cultures grown in the presence of 1 mM guanosine to induce the expression of the enzyme. The SHMT activity was found to be 3.2 U/mg of protein in strain 168 and <0.2 U/mg of protein in strain HH413. This indicates that the glyA gene actually encodes SHMT activity.

The derived amino acid sequence of yumD shows high amino acid sequence similarity to GMP reductase from E. coli (accession no. AAC73215) and to other putative GMP reductases and IMP dehydrogenases. The levels of GMP reductase were <0.03 U/mg of protein in HH355 (yumD) and 4.9 U/mg of protein in strain 168 grown in the presence of the inducer guanosine. This indicates that yumD encodes GMP reductase, and we suggest the new designation guaC.

The YqhZ primary structure has 40% amino acid sequence identity with the protein encoded by E. coli nusB (accession no. X00681). NusB has been shown to be involved in factor-dependent transcription termination in E. coli. An E. coli nusB mutant shows a reduced growth rate (19); however, this was not observed in a B. subtilis yqhZ mutant (see below). The derived amino acid sequence of folD has 52% amino acid sequence identity with the E. coli folD gene product (accession no. P24186), which encodes the bifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (3), which forms N¹⁰-formyl-tetrahydrofolate (N¹⁰-formyl-THFA), which is essential for de novo synthesis of IMP (Fig. 3). Another reaction in which N¹⁰-formyl-THFA is used is the synthesis of formylmethionyl tRNA, a reaction that is not essential for the growth of B. subtilis (1). To show that the folD gene actually encodes the enzyme catalyzing the last two steps in the synthesis of N¹⁰-formyl-THFA, conditions during which the gene was not expressed were studied. The folD gene is located downstream of yqhZ. In strain YQHZd, pMutin4 is integrated in yqhZ and expression of the downstream gene folD is driven by the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible P_spac promoter (20). The yqhZ::pMutin4 mutation was transformed into strain 168/pMAP65, which overproduces the LacI repressor protein encoded by the pMAP65 plasmid (9). A high level of LacI ensures that the P_spac promoter upstream of folD is almost completely shut down. The new strain ED448 (yqhZ::pMutin4 pMAP65) could grow in rich medium but not in minimal medium unless supplemented with IPTG or hypoxanthine (Table 7), indicating that the function ascribed to folD is correct. Growth was further increased when cells were grown in medium supplemented with Casamino Acids, indicating that protein synthesis can be increased despite the presumed lack of formylmethionyl tRNA in strain ED448 (Table 7). A low but significant level of N¹⁰-formyl-THFA synthetase activity has been measured in B. subtilis (22). N¹⁰-formyl-THFA synthetase catalyzes the synthesis of N¹⁰-formyl-THFA from THFA and formic acid. However, addition of formic acid to strain ED448 did not stimulate growth, indicating insufficient formation of N¹⁰-formyl-THFA from formic acid in the yqhZ mutant strain.

FIG. 3 — Map of metabolic pathways in *B. subtilis* that are regulated by PurR. The different enzymatic steps are represented by the corresponding gene designations. Gene designations in a large font and in boldface represent genes that are regulated by PurR, while gene designations in a small font represent genes that are not regulated by PurR. Abbreviations: GAR, phosphoribosylglycinamide; FGAR, phosphoribosylformylglycinamide; AICAR, phosphoribosylaminoimidazole carboxamide; FAICAR, phosphoribosylformamidoimidazole carboxamide; SAMP, adenylosuccinate. Gene designations: *purF*, glutamine PRPP amidotransferase; *purD*, phosphoribosylglycinamide synthetase; *purN*, THFA-dependent phosphoribosylglycinamide transformylases; *purQLS*, phosphoribosylformylglycinamidine synthetases I, II, and III; *purM*, phosphoribosylaminoimidazole synthetase; *purEK*, phosphoribosylaminoimidazole carboxylases I and II; *purC*, phosphoribosylaminoimidazolesuccinocarboxamide synthetase; *purB*, adenylosuccinate lyase; *purH*, phosphoribosylaminoimidazole carboxamide formyltransferase and IMP cyclohydrolase; *purA*, adenylosuccinate synthetase; *guaB*, IMP dehydrogenase; *guaA*, GMP synthetase; *apt*, adenine phosphoribosyltransferase; *hpt*, hypoxanthine-guanine phosphoribosyltransferase; *xpt*, xanthine phosphoribosyltransferase; *guaC*, GMP reductase; *ade*, adenine deaminase; *pbuG*, hypoxanthine-guanine permease; *pbuX*, xanthine permease; *pbuO*, guanine permease; *glyA*, SHMT; *folD*, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase. Dashed lines indicate multiple enzyme-catalyzed steps.

TABLE 7.

Effect of hypoxanthine and IPTG on growth of a folD mutant^a

Strain	Relevant genotype	Addition	Doubling time (min) in:
Strain	Relevant genotype	Addition	MM	Casamino Acids medium
ED448	folD	None	>600	>600
		IPTG^b	72	65
		Hypoxanthine	67	37
ED449	Wild type	Hypoxanthine	54	34

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Both strains contain plasmid pMAP65. Cells were grown in glucose minimal medium (MM) supplemented with neomycin, erythromycin, and lincomycin plus or minus 0.2% Casamino Acids (Casamino Acids medium).

Expression of folD was induced by addition of IPTG (0.2 mM).

The yebB gene is located close to the 5′ end of the pur operon, within a region that has previously been shown to contain the pbuG gene that encodes a hypoxanthine-guanine permease (14). Hypoxanthine uptake was measured in BFA2255 (yebB::pMutin1) and was found to be 0.1 U/mg of cell dry weight, compared to 2 U/mg of cell dry weight in strain 168. BFA2255, like a pbuG mutant strain (14), was found to be resistant to 0.5 mM azaguanine. These observations indicate that yebB and pbuG are the same gene, and we therefore suggest the original designation pbuG for yebB.

The YtiP sequence (432 amino acids) shows 47% amino acid sequence identity with the 440-amino-acid hypoxanthine-guanine permease PbuG. Strain BFA2025 (ytiP::pMutin1) was analyzed for its purine base uptake phenotype, and it was found that the mutant strain had a 50% reduction in guanine and hypoxanthine uptake compared to the wild type. This indicates that ytiP encodes a guanine-hypoxanthine permease. We suggest that the designation pbuO (purine base uptake, 6-oxopurine) replace the designation ytiP.

DISCUSSION

Based on the experimental results presented in this work, we were able to expand the B. subtilis PurR regulon with six mono- or dicistronic operons. The function and expression of the xpt-pbuX operon have been previously reported (2), while the functions of the genes yumD (guaC), yebB (pbuG), glyA, yqhZ-folD, and ytiP (pbuO) are described in this work. Two genes were shown to encode purine base permeases. yebB encodes a high-affinity hypoxanthine-guanine permease already known as pbuG (14). A pbuO (formerly ytiP) mutant was shown to be impaired in guanine and hypoxanthine uptake. The purine base concentration used in the uptake assay was low (1 μM). At this concentration, PbuG has been shown to be the major transport system for guanine because pbuG deficiency results in a low level of guanine uptake (14). The residual guanine uptake at 1 μM guanine in the pbuG mutant strain could be due to transport through PbuO. PbuG deficiency has no effect on the growth of a purine-requiring mutant strain when guanine or hypoxanthine is present at a concentration higher than 100 μM (14). Most likely, pbuO encodes a guanine-hypoxanthine permease working at purine concentrations higher than 100 μM.

Two genes, glyA and folD, encode enzymes involved in N¹⁰-formyl-THFA formation. Based on genetic data and on growth analysis of a glyA mutant, Dartois and coworkers suggested that glyA encodes SHMT (4). By measuring SHMT activity in a glyA knockout mutant, we have finally established the function of this gene in B. subtilis. folD was the only gene whose function was only indirectly demonstrated. The gene appears not to be essential as long as IMP can be synthesized from an external purine source. Finally, yumD (guaC) was identified as the gene encoding GMP reductase activity.

The previously identified PurR-regulated genes (pur operon and purA [17]) plus the newly identified ones allowed us to construct a map of the PurR-affected pathways in B. subtilis. In Fig. 3, it can be seen that the majority of the genes involved in purine base, purine nucleoside, and purine nucleoside monophosphate metabolism are regulated by PurR. Figure 3 also illustrates the three steps of THFA metabolism that are regulated by PurR. In E. coli, the formation of N⁵,N¹⁰-methylene-THFA is regulated by purine levels and PurR through the repression of glyA expression (18). However, folD in E. coli appears not to be controlled by PurR. Among all of the PurR-regulated genes, yqhZ, which encodes a potential NusB-like factor involved in transcription termination, is the only gene without an obvious role in purine metabolism.

We have shown that all of the B. subtilis genes and operons that have been experimentally demonstrated to be regulated by PurR are preceded by a palindromic sequence composed of two divergently oriented PurBoxes separated by 16 or 17 nucleotides. We have compared our data with previously obtained footprinting data (17) in which purified PurR protein was found to protect an extended region upstream of the pur operon, purA and the purR-yabJ operon. From this comparison, it is evident that the common dyad symmetry 5′-GAAC-N_(24–25)-GTTC-3′ motif identified by Shin and coworkers (17) is included in the tandem PurBox motif defined in this work (Fig. 1). Characteristic for the footprinting data are the large regions of 80 to 90 nucleotides that were protected by PurR protein. The extended protected regions reported by Shin and coworkers were found to be primarily on the 5′ side in relation to the two PurBoxes and the 5′-GAAC-N_(24–25)-GTTC-3′ motif. Analysis of the minimal regulatory sequence requirement for full PurR control of glyA expression revealed that no extended 5′ region relative to the tandem PurBox motif was required. This leads us to suggest that the binding of PurR to sequences upstream of the twin PurBox sequences, as demonstrated by previous in vitro footprinting experiments, most likely plays no role in vivo. The tandem PurBox motif may be located at various positions both up- or downstream of the transcriptional start site (Fig. 1). In the case of ytiP (pbuO), yumD (guaC), and purR, the PurBoxes are located close to or overlapping the sequence encoding the potential ribosome binding site. In the case of the pur operon, the xpt-pbuX operon, and yebB (pbuG), the PurBoxes are located 230 to 274 nucleotides upstream of the coding region of the first gene of the operon. This long distance is due to the presence of a long untranslated leader sequence that, in the case of the pur and xpt-pbuX operons, has been shown to be the site for the hypoxanthine-and-guanine-controlled regulatory mechanism. The PurBoxes in front of the pur operon, xpt-pbuX and pbuG, are located 4 (pur operon and pbuG) and 14 (xpt-pbuX) nucleotides upstream of the promoter −35 elements—distances that are consistent with the PurBoxes functioning as repressor binding sites. In the glyA regulatory region, the PurBoxes are located 35 nucleotides upstream of the −35 element. This may appear to be a rather long distance. However, as demonstrated by the published footprint analysis (17), PurR protects DNA sequences (approximately 20 nucleotides in length) located downstream of the 5′-GAAC-N_(24–25)-GTTC-3′ motif, which coincides with the PurBoxes. We speculate that PurR represses glyA transcription by first binding to the PurBoxes and then multimerizes along the DNA as suggested previously (7).

Addition of adenine to B. subtilis results in a drop in the cellular PRPP pool, thereby increasing the binding of PurR to its operator sequence. This results in an average of 2.5- to 3-fold repression of gene expression (Table 3) (10, 15). In contrast, addition of guanosine increases PRPP pools, resulting in decreased PurR binding and two- to threefold induction of gene expression (Table 7) (10, 15). Rappu and coworkers have suggested that stronger binding of PurR to operator DNA when the PRPP pool is low requires the yabJ gene product, and it was suggested that a possible function of YabJ is to interact with PurR to form a multimeric PurR structure. This would result in the binding and protection of the extended operator sequence by PurR observed in footprinting experiments. We investigated the effect of YabJ deficiency on the repression of expression of one of the novel PurR-controlled gene glyA and of purA, for which the repression was shown by Rappu and coworkers to be YabJ dependent. We were not able to detect any changes in either glyA or purA expression in a yabJ mutant strain compared to that in the wild type (Table 6). The two yabJ mutations, however, were not identical. Rappu and coworkers constructed a 39-amino-acid deletion of the YabJ (125 amino acids long) N-terminal end, whereas the mutation analyzed in this report was a 42-amino-acid deletion of the C-terminal end. Even though it appears unlikely that the repressor auxiliary function of YabJ may be dependent on the N-terminal part, this might be a possibility. Until this has been analyzed in more detail, the role of YabJ in the process of PurR-controlled gene expression remains questionable.

ACKNOWLEDGMENTS

We thank Jenny Steno Christensen and Kirsten Hansen for excellent technical assistance.

This work was supported by EU contract BIO2-CT95-0278 and by Danish Natural Science Research Council grant 9901855. This project also received financial support from the Novo Nordisk Foundation and from the Saxild Family Foundation.

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[B1] 1.Arnold H H. Initiation of protein synthesis in Bacillus subtilisin the presence of trimethoprim or aminopterin. Biochim Biophys Acta. 1977;476:76–87. doi: 10.1016/0005-2787(77)90287-8. [DOI] [PubMed] [Google Scholar]

[B2] 2.Christiansen L C, Schou S, Nygaard P, Saxild H H. Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuXoperon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism. J Bacteriol. 1997;179:2540–2550. doi: 10.1128/jb.179.8.2540-2550.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.D'Ari L, Rabinowitz J C. Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli. J Biol Chem. 1991;266:23953–23958. [PubMed] [Google Scholar]

[B4] 4.Dartois V, Liu J, Hoch J A. Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis. Mol Microbiol. 1997;25:39–51. doi: 10.1046/j.1365-2958.1997.4491805.x. [DOI] [PubMed] [Google Scholar]

[B5] 5.Ebbole D J, Zalkin H. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novopurine nucleotide biosynthesis. J Biol Chem. 1987;262:8274–8287. [PubMed] [Google Scholar]

[B6] 6.Kilstrup M, Jessing S G, Wichmand-Jørgensen S B, Madsen M, Nilsson D. Activation control of pur gene expression in Lactococcus lactis: proposal for a consensus activator binding sequence based on deletion analysis and site-directed mutagenesis of purC and purDpromoter regions. J Bacteriol. 1998;180:3900–3906. doi: 10.1128/jb.180.15.3900-3906.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Kilstrup M, Martinussen J. A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis. J Bacteriol. 1998;180:3907–3916. doi: 10.1128/jb.180.15.3907-3916.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Kunst F, Ogasawara N, Moszer I, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997;390:249–256. doi: 10.1038/36786. [DOI] [PubMed] [Google Scholar]

[B9] 9.Petit M A, Dervyn E, Rose M, Entian K D, McGovern S, Ehrlich S D, Bruand C. PcrA is an essential DNA helicase of Bacillus subtilisfulfilling functions both in repair and rolling-circle replication. Mol Microbiol. 1998;29:261–273. doi: 10.1046/j.1365-2958.1998.00927.x. [DOI] [PubMed] [Google Scholar]

[B10] 10.Rappu P, Shin B S, Zalkin H, Mantsala P. A role for a highly conserved protein of unknown function in regulation of Bacillus subtilis purAby the purine repressor. J Bacteriol. 1999;181:3810–3815. doi: 10.1128/jb.181.12.3810-3815.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Rolfes R J, Zalkin H. Escherichia coli gene purRencoding a repressor protein for purine nucleotide synthesis. J Biol Chem. 1988;263:19653–19661. [PubMed] [Google Scholar]

[B12] 12.Saxild H H, Andersen L N, Hammer K. dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein. J Bacteriol. 1996;178:424–434. doi: 10.1128/jb.178.2.424-434.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Saxild H H, Jacobsen J H, Nygaard P. Functional analysis of the Bacillus subtilis purTgene encoding formate-dependent glycinamide ribonucleotide transformylase. Microbiology. 1995;141:2211–2218. doi: 10.1099/13500872-141-9-2211. [DOI] [PubMed] [Google Scholar]

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PERMALINK

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

Katja Brunstedt

Karin I Nielsen

Hanne Jarmer

Per Nygaard

Abstract