Vol. 176, No. 2
JOURNAL OF BAC-ERIOLOGY, Jan. 1994, p. 276-283
0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Isolation and Characterization of Bacillus subtilis Genomic lacZ Fusions Induced during Partial Purine Starvation HANS H. SAXILD,* CHRISTINA L. JENSEN, PETER HUBRECHTS, AND KARIN HAMMER Department of Microbiology, The Technical University of Denmark DK-2800 Lyngby, Denmark Received 1 September 1993/Accepted 11 November 1993
Random genomic Bacilus subtilis lacZ fusions were screened in order to identify the possible existence of regulons responding to the stimuli generated by partial purine starvation. A leaky pur mutation (purL8) was isolated and used to generate the partial purine starvation conditions in the host strain used for screening. On the basis of their induction during partial purine starvation, seven genomic lacZ fusions were isolated. None of the fusions map in loci previously reported to contain purine-regulated genes. One fusion maps very close to the citB locus and may very well be a citB fusion. The fusions were divided into two types on the basis of their response to complete starvation for either ATP or GTP or both components at the same time. Except for one, type 2 fusions were induced by specific starvation for ATP and by simultaneous starvation for ATP and GTP, but not by specific GTP starvation in a gua strain or by GTP starvation induced by the addition of decoyinine. Type 1 fusions were equally well induced by all three kinds of purine starvation including GTP starvation induced by decoyinine. Further subdivisions of the fusions were obtained on the basis of their responses to the spoOA gene product. A total of five fusions showed that spoOA affected expression. One class was unable to induce lacZ expression in the absence of the spoOA gene product, whereas the other class had increased lacZ expression during partial purine starvation in a spoOA background.
(7, 34). By correlating nucleotide pools and enzyme levels, it has been suggested that the purA and guaB expressions are regulated by the GTP/ATP pool ratio (34). Finally, the nucleotide sequence of the 5' end of the guaA gene reveals an untranslated leader sequence identical in structure to the one found in the pur operon. This could suggest a common guanine-responding control mechanism for transcription termination in B. subtilis (25). In B. subtilis, purine regulation is of special interest, since the initiation of sporulation and the GTP pool size is coupled via a mechanism that is not yet known. Massive sporulation can be induced in B. subtilis by starving leaky purine auxotrophs for purines or treating cells with drugs inhibiting GTP synthesis even in the presence of excess nitrogen, phosphate, and carbon (10, 24). On the other hand, when sporulation is initiated by depletion of the above-mentioned nutrients, a drop in the GTP pool is always observed (18). Whether the GTP pool size is a direct intracellular effector for sporulation initiation or merely a secondary indicator of other processes related to sporulation is not known. On the basis of the data summarized above, our working hypothesis is that one or more global purine-regulated control systems for gene expression exist in B. subtilis. To further characterize these control systems, we attempted, as a first step, to identify new genes subjected to purine regulation. As a genetic tool, we used a series of integrational plasmids, which, upon integration into the chromosome, are capable of generating random fusions to the E. coli lacZ gene (8). A leaky pur mutant was isolated and used for the screening of the lacZ fusions. We describe the isolation and physiological and genetic characterization of seven genomic lacZ fusions with altered expression during partial purine starvation.
Purine compounds play a central role in cellular metabolism. In addition to serving as substrates for nucleic acid synthesis, they are essential compounds in energy metabolism and protein synthesis. They are substrates for many enzyme-catalyzed reactions including, for example, the biosynthesis of coenzymes and vitamins. Purine compounds (nucleobases and nucleotides) exert many regulatory functions in the cell. They act as modulators of many allosterically regulated enzymes and as coeffectors involved in the regulation of gene expression (in
procaryotes). The direct role of purines in gene regulation has been demonstrated for Escherichia coli. Rolfes and Zalkin (29) have characterized a gene, purR, encoding a repressor protein for which the purine bases hypoxanthine and guanine have been identified as the true corepressors (21). The PurR regulon consists of all genes encoding enzymes required for the biosynthesis of AMP and GMP except the purA gene (13, 14, 20, 29). The PurR repressor has also been shown to control the expression of the purR,pyrC, pyrD, cod, glyA, and gcv genes (1, 29, 37, 40, 41). Expression of the purA gene has been shown to be repressible by adenine; however, it is not regulated by PurR. This indicates the operation of a second purine-dependent control mechanism in E. coli (19). In Bacillus subtilis, purines have been shown to be involved in the regulation of the pur operon as well as the purA and guaB genes. The pur operon consists of 12 genes encoding all of the enzymes involved in the biosynthesis of IMP (7). The levels of the enzymes encoded by the pur operon are repressible by adenine and guanine compounds. Their expression is transcriptionally regulated by two separate mechanisms. One is a repressor mechanism for which an adenine nucleotide has been suggested as the coeffector, and the second is a premature termination mechanism regulated by a guanine compound
MATERIALS AND METHODS
* Corresponding author. Mailing address: Department of Microbiology, The Technical University of Denmark, Building 221, DK-2800 Lyngby, Denmark. Phone: 45 93 12 22 local 2495. Fax: 45 88 26 60.
Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this work are listed in Table 1. E. coli
Electronic mail address:
[email protected].
276
PURINE-REGULATED GENES IN B. SUBTILIS
VOL. 176, 1994
277
TABLE 1. Bacterial strains and plasmidsa Strain or plasmid
B. subtilis 168
1S53 QB917 ED156 ED159 ED250 ED280 HH9 HH41 HH26 HH42 HH69 HH71 HH74 HH76 HH82 HH123
HH(26-82).spod ED159.lacZ-(26-82) ED250.lacZ-(26-82) ED280.lacZ-(26-82) HH123.lacZ-(26-82) E. coli MT102
Plasmids pSGMU32 pSGMU37 pSGMU38
Source
Genotype or phenotype
trpC2 spoOAA677 trpC2 hisAl thr-5 trpC2 hisAl purF6 trpC2 hisAl purM1 trpC2 hisA1 thr-5 purA2 trpC2 hisAl guaBI trpC2 purL8 trpC2 purL8 trpC2 purL8 prg-26::lacZ (cat) trpC2 purL8 prg-42::lacZ (cat) trpC2 purL8 prg-69::1acZ (cat) trpC2 purL8 prg-71::lacZ (cat) trpC2 purL8 prg-74::lacZ (cat) trpC2 purL8 prg-76::lacZ (cat) trpC2 purL8 prg-82::lacZ (cat) trpC2 hisAl purL8 prg-(26-82)::lacZ (cat) spoOAA677 trpC2 hisAl purMI prg-(26-82)::lacZ (cat) trpC2 hisAl thr-5 purA2 prg-(26-82)::1acZ (cat) trpC2 hisAl guaBi prg-(26-82)::1acZ (cat) trpC2 hisAl prg-(26-82)::1acZ (cat)
C. Anagnostopoulos BGSCb R. A. Dedonder (BGSC) H. H. Saxild and P. Nygaard H. H. Saxild and P. Nygaard H. H. Saxild and P. Nygaard H. H. Saxild and P. Nygaard This work Tf of ED156 by HH9 (His' and PurF+) This work; EcoRV library in pSGMU32 This work; HindIII library in pSGMU37 This work; PstI library in pSGMU38 This work; PstI library in pSGMU38 This work; PstI library in pSGMU38 This work; PstI library in pSGMU38 This work; PstI library in pSGMU38 ED361 (Tf of QB917 by 168 [Thr+]); P. Nygaard Tf of HH(26-82) by 1S53 (Trp+) Tde of ED159 by HH(26-82); Cmr Td of ED250 by HH(26-82); Cmr Td of ED280 by HH(26-82); Cmr Td of HH123 by HH(26-82); Cmr
F-, araDJ39 A(argF-leu)7696 A(lac)X74 galU, galK hsdR2 (r-m+), mcrBl rpsL (Strr)
MC1061; laboratory stock
Apr Cmr (transcriptional lacZ fusions) Apr Cmr (translational lacZ fusions) Apr Cmr (transcriptional lacZ fusions)
J. Errington J. Errington J. Errington
a The Dedonder mapping kit was from R. A. Dedonder (BGSC); the Tn917 mapping kit was from M. A. Vandeyar and S. A. Zahler (BGSC). b BGSC, Bacillus Genetic Stock Center. c Tf, transformation of the first stain with DNA from the second as described in Materials and Methods. d Isogenic series of strains containing the -42, -69, -71, -74, -76, and -82 fusions, respectively. e Td, transduction of the first strain with AR9 phages propagated on the second as described in Materials and Methods.
prg-26::lacZ,
MT102 was cultured in L broth (Difco Laboratories, Detroit, Mich.). For enzyme assays, B. subtilis strains were grown in Spizizen minimal salt medium (31) supplemented with 1 ,ug of thiamine hydrochloride per ml, 50 ,ug of L-tryptophan per ml, 0.2% L-glutamate, 0.4% glucose, and 100 ,iM the stated purine compounds. Additional amino acids required by auxotropic strains were added at 50-,ug/ml concentrations. Plate screenings for ,B-galactosidase activity were done on Spizizen minimal medium solidified with 1.5% agar. For induction of sporulation, Schaeffer's nutrient broth sporulation medium was used (35). Selection for antibiotic-resistant B. subtilis transductants and transformants was performed on L broth agar plates containing the relevant antibiotics. For selection of antibiotic resistance, the following concentrations were used: ampicillin, 100 ,ug/ml; chloramphenicol, 5 pug/ml; and erythromycin and lincomycin, 1 and 25 ,ug/ml, respectively. Decoyinine (Upjohn Company, through T. Atlung) was dissolved in 1 M KOH immediately before use and added at a final concentration of 500 ,ug/ml. The total viable cell titer was determined by plating dilutions on L broth agar plates, and spore titers were determined by heating 200-,lI samples of cell cultures to 75°C for 20 min prior to plating. Transformation and transduction. B. subtilis chromosomal DNA was prepared according to the method described by Saxild and Nygaard (32). Large-scale plasmid preparations from E. coli were prepared from 200-ml L broth cultures by the alkaline-sodium dodecyl sulfate method (3). Transformation of
E. coli (12) and B. subtilis (32) and AR9-mediated transduction in B. subtilis have been described previously (32).
Chemicals, isotopes, and enzymes. Fine chemicals and antibiotics were from Sigma Chemical Company, St. Louis, Mo. Carrier-free [32P]phosphoric acid was from Fors0gsanlkg, Ris0, Denmark. Restriction endonucleases and T4 ligase were obtained from New England Biolabs, Inc., Beverly, Mass. Digestion and ligatioil of DNA were done as recommended by the supplier. ATP and GTP pool determinations. [32P]phosphoric acid (0.8 MBq) was added to 2 ml of B. subtilis HH41 (purL8) growing exponentially in low-phosphate medium (26). At an optical density at 450 nm (OD450) of 0.8, samples of 200 ,ul were extracted with ice-cold formic acid, and the radiolabeled nucleoside triphosphates were separated by two-dimensional chromatography on polyethyleneimine-impregnated cellulose on plastic sheets (16). After separation of the nucleotides, the ATP and GTP spots were cut out, and the radioactivity was determined by liquid scintillation counting. Plate screening for 13-galactosidase activities and j8-galactosidase assays. Detection of ,B-galactosidase activity on agar plates was done by spraying colonies with a 10-mg/ml solution of 4-methylumbelliferyl-,-D-galactoside (MUG) in dimethyl sulfoxide. After 10 to 20 min at 37°C, the colonies were exposed to UV light (312 nm), and light-blue fluorescence was detected. For assaying 3-galactosidase activity in liquid cultures, a previously described method was used (28). Essen-
278
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SAXILD ET AL.
tially, 1.5 ml of an exponentially growing or starving culture harvested in microcentrifuge tubes and immediately froat 70°C. Cells were thawed by adding 1 ml of Z buffer (23) containing 500 ,ug of lysozyme per ml. Lysis was completed after 5 min at 37°C. Tubes were placed at 28°C, and the 3-galactosidase assay was started by adding 200 ,ul of orthonitrophenol in Z buffer (4 mg/ml). After a sufficient incubation period, 0.5 ml of 1 M Na2CO3 was added and the tubes were centrifuged for 5 min. Finally, the OD420 in the supernatant was measured. ,3-Galactosidase activities are presented as Miller units (23), which are computed as follows: (1,700 x OD420) x (T x V x OD450) -1, where T = reaction time in minutes, V = culture volume in milliliters, and OD450 = OD of the culture at the sample time. Construction of B. subtilis genome libraries in the pSGMU lacZ fusion vectors. Approximately 1.5 ,ug of chromosomal DNA from B. subtilis 168 was digested to completion with an appropriate enzyme and ligated with approximately 0.25 ,ug of pSGMU plasmid linearized with the same enzyme or an enzyme creating compatible ends. Three libraries were constructed. EcoRV DNA fragments were ligated into the SmaI site of pSGMU32, HindlIl fragments were ligated into the HindIII site of pSGMU37, and PstI fragments were ligated into the PstI site of pSGMU38. The ligation mixtures were then transformed into E. coli MT102, selecting for Apr. A total of 8 X 103 to 12 x 103 Apr colonies derived from each ligation mixture were pooled, and the plasmid content was extracted. EMS mutagenesis and penicillin selection. Ethyl methanesulfonate (EMS) mutagenesis of B. subtilis 168 was performed essentially as described by Miller (22). All incubations were done at 37°C. A total of 10 ml of exponentially growing B. subtilis cells at an OD450 of 1.0 were harvested, resuspended in 10 ml of SC buffer (0.15 M NaCl, 0.01 M sodium citrate [pH 7.0]), and the 125 ,ul of EMS was added. The culture was incubated for a time period that resulted in approximately 90% killing. Phenotypic expression was allowed by growing the culture for approximately 10 generations in minimal medium supplemented with 100 ,uM hypoxanthine. The culture was diluted to an OD450 of 0.05 in hypoxanthine-supplemented minimal medium and was grown exponentially to an OD450 of 0.5, after which the cells were harvested, washed with 0.9% NaCl, and resuspended in minimal medium lacking purine. After 15 min of further incubation, 2,000 U of penicillin G (Sigma) per ml of culture was added, and incubation continued for 2 h. Penicillin was removed by washing the cells four times with 10 ml of 0.9% NaCl. Finally, the cells were resuspended in sterile water, and 10-fold dilutions were plated on minimal agar plates containing hypoxanthine. After 48 h of incubation, colonies were replicated onto minimal plates lacking purine and clones requiring hypoxanthine for growth (approximately 0.5 to 1%) were picked and purified. was zen
PRPP
-
steps
purMQ
Characterization of the purL8 mutation. Purine auxotrophic mutants of strain 168 were made by the procedure described in Materials and Methods. In one selection, 11 purine-requiring mutants were isolated. One of these showed a leaky phenotype. In a transformation analysis using purified B. subtilis pur DNA fragments, it was found that a fragment containing the purL gene (Fig. 1) (nucleotides 5088 to 6982 in reference 7) could rescue (by recombination) the leaky Pur- phenotype (data not shown). The mutation was moved by transformation into strain ED156, creating strain HH41 (trpC2purL8). Strain HH41 had a decreased growth rate and reduced ATP and GTP pools and
-
V
ADP
IP
GDP
5steps
AMP--- SAMP
IMP
-
XMP
--
guaB
purA
GMP
Gua
Hyp
Ado
FIG. 1. Outline of the purine biosynthetic pathway and the purine interconversion reactions in B. subtilis. Only relevant enzymatic steps are shown. The enzymatic steps are indicated by their gene symbols. purLQ, formylglycineamide ribonucleotid amidotransferase; purM, aminoimidazole ribonucleotid synthetase; purA, adenylosuccinate synthetase; guaB, IMP dehydrogenase; PRPP, phosphoribosyl pyrophosphate; Ade, adenine; Hyp, hypoxanthine; Gua, guanine.
produced spores during growth in minimal medium lacking purine (Table 2). Isolation of genomic lacZ fusions having altered expression during partial purine starvation. The B. subtilis genome was screened for genes induced during partial purine starvation by means of integrative plasmids containing a promoterless lacZ reporter gene preceded by the spoIL4A ribosome binding site (in pSGMU32 and pSGMU38) or lacking both transcriptional and translational start signals (in pSGMU37) (8). The pSGMU-derived plasmids are able to create chromosomal lacZ fusions by integrating at the site of homology given by the cloned insert (Fig. 2). DNA from three plasmid pools (see Materials and Methods) was introduced into strain HH41 (purL8), selecting for Cmr on LB medium. Approximately 2,000 Cmr transformants derived from each plasmid pool were screened by replica plating onto minimal agar plates with or without purine. To obtain equal colony sizes on both sets of plates, the purine-containing plates were incubated for 24 h at room temperature and then for 24 h at 37°C, while plates lacking purine were incubated for 48 h at 37°C. A total of 10 to TABLE 2. Effect of the presence of purine in the growth medium on purine ribonucleoside triphosphate pools and spore production in a B. subtilis wild-type strain and a purL8 derivative" Ptririne
(relevant
genotyp)
RESULTS
GTP
ATP
Puie
Padded
HH123 (wild Hypoxanthine None type) HH41 Hypoxanthine None (purL8)
bingNucleotide
time (min)b 41 41 41 240
pools AT'PGP 100 100 100 30
100 100 100 50
Heat-
Viable
res'istant
cells/mld spores/ mle 8.2 7.5 6.8 1.3
x x x x
107 107 107
21 24 19
108 5.7 x 105
a Cells were grown in minimal medium in the presence or absence of 100 ,M hypoxanthine. Triplicate determinations were identical. Nucleotide pools are given as percentages of the values found in the wild-type strain grown without hypoxanthine (ATP, 3.5 nmol/mg [dry weight]; GTP, 1.0 nmol/mg [dry weight]). Duplicate determinations varied by less than 5%. d Cells were grown exponentially for approximately 10 generations. At an OD450 of 1.0, the total viable cell and heat-resistant spore titers were determined as described in Materials and Methods.
PURINE-REGULATED GENES IN B. SUBTILIS
VOL. 176, 1994
TABLE 3. Expression of the B. subtilis genomic prg::lacZ fusions during exponential growtha P-Galactosidase activity (Miller units)b Relevant genotype Strain
cat
bla
sts genome libraries pSGMU32, 37 and 38
in
acZ
x_
P777777777777
B.subtilis
laZ
cat
279
bla
chromosome
ori
FIG. 2. The principle for generation of chromosomal lacZ fusions in B. subtilis using the integrable pSGMU plasmid vectors. Open bars, pSGMU plasmid DNA; black bars, plasmid-encoded genes; hatched bars, B. subtilis genome; shaded bar, cloned B. subtilis chromosomal DNA. lacZ, 3-galactosidase; bla, 3-lactamase; cat, chloramphenicol acetyltransferase; ori, pUC replication origin.
15% of the colonies showed P-galactosidase activity when sprayed with MUG. Clones showing increased activity on purine-free medium were picked from the LB master plate and purified. A total of seven candidates were isolated by this procedure. The fusion prg-26::1acZ (prg stands for purineresponding gene regulation) is a transcriptional fusion isolated from a lacZ fusion library of random B. subtilis EcoRV fragments cloned into the SmaI site of pSGMU32.prg-42::1acZ is a translational fusion isolated from a library of HindlIl fragments ligated into the HindlIl site of pSGMU37. prg-69:: lacZ, prg-71::lacZ, prg-74::lacZ, prg-76::lacZ, and prg-82::1acZ are transcriptional fusions isolated from a library of PstI fragments cloned into the PstI site of pSGMU38. Phenotypes. None of the prg::lacZ insertions caused any detectable phenotype, except for 3-galactosidase activity. No changes in growth, sporulation, or transformability were observed (data not shown). Integration of plasmids into the chromosome by a single crossover event usually does not lead to gene inactivation. Only if the cloned insert constitutes an internal part of a transcriptional unit does the insertion result in gene disruption. Expression of prg::lacZ fusions. To quantify the expression of the prg::lacZ fusions during partial purine starvation, cells were grown exponentially for many generations (approximately 10) by two consecutive dilutions in liquid minimal medium in the absence or presence of purine. The regulatory pattern of the prg::lacZ fusions is listed in Table 3. Expression of all of the lacZ fusions was increased 3- to 38-fold when cells were grown without purine added to the medium, i.e., under purinelimiting conditions. In the experiment shown in Table 3, hypoxanthine was used as a purine source. The same regulatory pattern was observed when adenine or guanine was substituted for hypoxanthine (data not shown). In another experiment, the different prg::lacZ fusion strains were grown in minimal medium containing 20 ,uM hypoxanthine, which supports normal growth (40 min of generation time), to an OD450 of approximately 0.5. After exhaustion of the purine source, the strains grew at a decreased rate (240-min generation time; see Table 2). The differential rate of 3-galactosidase synthesis was measured before and for 2 h after exhaustion of hypoxan-
HH41 HH41.spo HH26 HH26.spo HH42 HH42.spo HH69 HH69.spo HH71 HH71.spo HH74 HH74.spo HH76 HH76.spo HH82 HH82.spo
purL8 purL8 spoOA purL8 prg-26::lacZ purL8 prg-26::lacZ spoOA purL8 prg-42::1acZ purL8 prg-42::1acZ spoOA purL8 prg-69::lacZ purL8 prg-69::1acZ spoOA purL8 prg-71::lacZ purL8 prg-71::lacZ spoOA purL8 prg-74::1acZ purL8 prg-74::lacZ spoOA purL8 prg-76::lacZ purL8 prg-76::lacZ spoOA purL8 prg-82::lacZ purL8 prg-82::lacZ spoOA
+Hyp
- Hyp
0.1 0.1
0.1 (1) 0.1 (1) 2.5 (5.0) 0.9 (1.5) 10.6 (11.7) 2.1 (1.9) 15.0 (37.5) 17.0 (42.5) 8.7 (21.8) 18.0 (6.0) 20.6 (9.4) 0.8 (0.4) 5.8 (2.9) 17.0 (5.5) 39.4 (14.6) 30.0 (13.6)
0.5 0.6 0.9 1.1 0.4 0.4 0.4 3.0 2.2 2.2 2.0 3.1 2.7 2.2
a Response to partial purine starvation in a leaky purine-requiringpurL8 strain and in a purL8 spoOA derivative. Bacterial strains were grown in minimal medium with (+) or without (-) addition of hypoxanthine (Hyp). b Means of three experiments. Numbers in parentheses indicate enzyme levels related to those found when cells were grown in medium supplemented with hypoxanthine.
thine. All of the prg::lacZ fusions were immediately induced after the shift from normal to partially purine-starved growth conditions (data not shown). Expression during starvation for different purine compounds. The response of the prg::lacZ fusions to starvation for specific purine compounds was analyzed by transducing the prg::lacZ fusions into three purine auxotrophic strains having defects in different parts of the purine biosynthetic pathway but otherwise isogenic (Fig. 1). Strain ED280 (guaB) is IMP dehydrogenase negative and requires guanine for growth. IMP dehydrogenase catalyzes the first step in the guanine-specific pathway leading from IMP to GMP. Strain ED250 (purA) produces a defective sAMP synthetase enzyme and requires adenine for growth. Adenylosuccinate (sAMP) synthetase catalyzes the first step of the adenine-specific pathway leading from IMP to AMP. Finally, strain ED159 (purM) is defective in the fifth step (catalyzed by AIR synthetase) of the purine de novo pathway. The de novo pathway leads from phosphoribosyl pyrophosphate to IMP (Fig. 1). The specific activities of P-galactosidase in both growing and starving cultures of the purine auxotrophic strains carrying the prg::lacZ fusions were determined (Table 4). They were all strongly induced by general starvation for both GTP and ATP (strain ED159 [purM]), the fold of induction being equal to or greater than those found in the leaky purL8 mutant in which the fusions were originally screened (Table 3). During specific starvation for ATP (strain ED250 [purA]), prg-26::1acZ, -42, -69, and -76 were fully induced, whereas prg-71::lacZ, -74, and -82 were only partially induced (to 30 to 50% of the levels obtained by general purine starvation). On the other hand, during starvation for GTP (in strain ED280 [guaB]), only fusionprg-76::lacZ was fully induced, while all other fusions were either uninduced (prg-26::1acZ, -42, -69, and -82) or showed only weak (prg-71::1acZ) to moderate (prg-74::1acZ) induction. Expression in a Pur+ background. The seven lacZ fusions were transduced to the wild-type strain HH123 (trpC2 hisAl) to investigate the purine response in a Pur+ background.
280
SAXILD ET AL.
J. BACTERIOL.
TABLE 4. Expression of the genomic prg::lacZ fusions during complete purine starvation of purine auxotrophic B. subtilis derivatives blocked in different steps of the purine
:B 15
biosynthetic pathway' 1:
Relative 13-galactosidase activity of the following strains after
2 h of starvation for the following purine compoundsb:
prg::lacZ fusion
ED280
None prg-26::lacZ
prg-42::1acZ prg-69::lacZ
prg-71::1acZ prg-74::lacZ
prg-76::1acZ prg-82::lacZ a
Strains
(guaB)
for
ED250
(purA)
for
ED159
(purM)
- 5 for
guanine
adenine
hypoxanthine
3.0 (0.1) 1.4 (1.2) 1.8 (4.8) 1.3 (0.7) 4.3 (0.6) 3.0 (2.4) 3.7 (2.5) 1.7 (0.9)
1.0 (0.1) 4.1 (1.1) 21.4 (0.7) 53.0 (1.3) 14.6 (2.4) 3.6 (3.7) 5.5 (2.9) 13.8 (2.1)
2.0 (0.1) 4.7 (0.7) 23.8 (1.3) 60.0 (0.4) 41.0 (1.0) 8.2 (2.9) 4.6 (3.6) 26.4 (2.5)
were grown
with 100
,uM the required purine compound. At
-10
a
0.1-I -0 -1 0 1 2 3 4 5 I
0
0
15 101
Ia
an
-1
OD450 of 0.7, cells were harvested and resuspended in minimal medium lacking
purine, and incubation was continued for 2 h, after which P-galactosidase activity was determined. b The relative 13-galactosidase activity is given as the ratio between the specific activity after 2 h of purine starvation and the specific activity in the culture before removal of purine. B. subtilis ED280 (guaB) requires guanine, ED250 (purA) requires adenine, and ED159 (purM) requires hypoxanthine for growth. Figures in parentheses are the specific ,-galactosidase activities in strains growing in the presence of purine.
When the Pur+ derivatives were grown in the presence of hypoxanthine, their enzyme levels were all identical to those found in the purL8 background (Table 3). However, except for the prg-69::lacZ fusion, none of the fusions was induced when cells were grown in the absence of purine (data not shown). These data indicate that the fusions, except for prg-69::1acZ, are induced only when purine nucleotide pools drop beneath the normal wild-type levels. Whether the regulatory mechanism relates directly or indirectly to the purine nucleotide pools cannot be judged from this experiment. The one exception, prg-69::1acZ, showed a 5.5-fold-increased level of expression in medium lacking purine (from 0.6 to 3.3 Miller units). Effect of SpoOA and induction by decoyinine and in sporulation medium. When a strain carrying the purL8 mutation is growing in the absence of exogenous purines, it contains a reduced cellular concentration of GTP, as shown in Table 2. Since a drop in the cellular concentration of GTP is known to initiate sporulation (10) and since the indicator strain HH41 spontaneously produces spores during growth in the absence of purines, we wanted to determine whether the expression of any of the fusions was affected by the spoOA gene product. This was done by introducing the spoOAA 677 deletion mutation into all of the prg::lacZ fusion strains by DNA-mediated transformation. The purine responses of the prg::lacZ fusions in isogenic spoOA and spoOA+ derivatives are listed in Table 3. Only the prg-69::lacZ and prg-82::lacZ fusions were completely unaffected by SpoOA. The rest could be divided into two groups on the basis of their expression patterns in the presence of the spoOA mutation. First, the prg-26::1acZ, prg-42::lacZ, and prg-74::1acZ fusions could not be induced by purine limitation in a spoOA background; however, they still had normal expression levels in the presence of purines. Second, the spoOA derivatives of the prg-71::lacZ and prg-76::1acZ fusion strains had increased expression compared with the Spo+ derivatives. prg-71::lacZ was derepressed both in the presence and in the absence of purine, resulting in less-pronounced purine regulation. prg-76::lacZ showed derepression only during purine limitation, resulting in an increased fold of induction. Three independent spo mutant transformants of each fusion strain
0
1
2
3
4
5
nlme (h) FIG. 3. Effect of decoyinine on growth and on the induction of the prg-71::1acZ, -74, -76, and -82 fusions. prg-71::lacZ (A), prg-74::1acZ (B),prg-76::1acZ (C), and prg-82::1acZ (D) are shown. Squares, optical density (OD450); Triangles, 1-galactosidase activity. Open symbols, data from untreated cultures; closed symbols, data from cultures treated with decoyinine. to corresponds to the time of addition of decoyinine. ,3-Galactosidase activity was measured as described in Materials and Methods. Note that the scale for 13-galactosidase activity in panel D is different.
have been isolated and examined, and the
same
regulatory
pattern was observed for all three. This experiment shows that
the screening procedure indeed detects fusions, which are influenced by the action of the spoOA gene product. It also indicates that some kind of sporulation signal is present in the purL8 strain during partial purine starvation, even though the cultures appear to grow exponentially with a defined growth rate.
To further characterize the effects of sporulation signals on the prg::lacZ fusion expression, massive sporulation was induced by adding the GMP synthetase inhibitor decoyinine to the series of isogenic wild-type strains containing the different fusions [the strains HH123.1acZ-(26-82)]. At an OD450 of 0.7, an exponentially growing culture was divided into two halves, and decoyinine (final concentration, 500 ,ug/ml) was added to one of them. At appropriate time intervals after decoyinine addition, samples were withdrawn and the specific activity of I-galactosidase was determined. Figure 3 summarizes the results of this experiment. The expression of four of the fusions (prg-71::lacZ, -74, -76, and -82) was clearly induced at the onset of sporulation (time to in Fig. 3). When guanine was added to the growth medium, no induction by decoyinine was observed (data not shown). The expression of the prg-26::lacZ, -42, and -69 fusions was unaffected. Sporulation of the strains HH123.lacZ-(26-82) was also induced by outgrowth in Schaeffer's nutrient sporulation medium. Samples for determination of P-galactosidase activity were taken before and for 2 h after growth had stopped. Among the SpoOA-affected fusions, only prg-42 and -71 were induced under these circumstances. prg-26, -74, and -76 and the SpoOA-independent fusions prg-69 and -82 were not induced (data not given). Map positions. To localize the insertion points of the fusions, the cointegrated cat gene (conferring chloramphenicol resistance) (Fig. 2) was used as a selectable marker (11). AR9
PURINE-REGULATED GENES IN B. SUBTILIS
VOL. 176, 1994
TABLE 5. Cotransduction between prg::lacZ (cat) insertions and auxotrophic markers and silent Tn9l 7 insertions prg::lacZ (cat) insertion
prg-26::lacZ prg-42::lacZ prg-69::lacZ prg-71::lacZ
(0)
(cat)
zej-82::Tn917 (18) and thyA (22) thr (53), cysB (56), and hisA (3) zhi-83::Tn917 (98) and thr (56) metC (37), proAB (38), and glyB (2) zif-85::Tn917 (94), purA (48), and ahrA (56) zif-85::Tn917 (35) zej-82::Tn917 (22), thyA (68), and citB (99)
176 288 280 115 342
(cat)
(cat) (cat) prg-74::lacZ (cat) prg-76::1acZ (cat)
NDa 173
prg-82::lacZ (cat) a
Cotransducible marker(s) (% cotransduction)
Location
ND, not determined.
lysates propagated on the fusion strains were transduced into the strains from the Tn917 mapping kit described by Vandeyar and Zahler (39), selecting for Cmr. The transductants were screened for simultaneous loss of the Tn917 element (Err). All fusions except prg-42::lacZ and prg-71::lacZ were localized by this procedure. The prg-42::1acZ and prg-71::1acZ fusion points were found by transducing the different Dedonder mapping strains (5) to Cmr and screening for simultaneous rescue of the recipient markers. The cotransduction frequencies between the fusion points and neighboring markers are listed in Table 5. All positions except prg-76::lacZ have been ordered relative to nearby markers by three-factor crosses (data not shown). The localization of the fusions on the genetic map is shown in Fig. 4. It appears that the fusion points are scattered on the chromosome. The three-factor cross involving prg-71::lacZ, metC, and proAB clearly indicates that the fusion is placed between metC and proAB with a cotransduction frequency of 37 to 38% to both markers (data not shown). The data therefore suggest that metC and proAB are not so closely linked as previously reported (27) (they have both been assigned to 115° on the linkage map). DISCUSSION To identify the existence of regulons in B. subtilis responding to stimuli generated by partial starvation for purines, the
prg-74::1acZ prg-76:-:lacZ
zf-85::\
_Tn917\ prg-42::Bac prg-69::1acZ
thr
Tn917
t0+o
~~~~~~~proAB /
\ \
~~~~~~metC prg-71::lacZ
prg-26::1acZ
prg-82::1acZ
FIG. 4. Map positions of the prg::lacZ integrations. The indicated positions are based on the data from Table 5 and on the frequency of recombinant groups in three-factor crosses.
281
following strategy was used. Random lacZ fusions into the chromosome of B. subtilis were obtained by constructing in E. coli libraries of the B. subtilis genome in the pSGMU integration plasmid vectors (8) and then by transformation into B. subtilis and selection for integration. Although the Tn917::lacZ elements have been widely used in similar in vivo screening procedures (2, 4, 15), we chose the plasmid system because of the potentially nonmutagenic nature of the single crossover integration event. Thus, this enables the isolation of fusions to genes essential for growth and viability. A leaky pur mutation (purL8) was isolated and used to generate the partial purine starvation conditions in the host cell used for screening of the chromosomal lacZ fusions generated by the insertion events. When grown without added purine, strains containing fusions showing induced lacZ expression were obtained with a frequency of approximately 1/1,000 Cmr B. subtilis transformants. Seven lacZ fusions were subjected to further analysis with respect to map positions, specificity of the purines involved in the inducing signal (including the induction pattern in the presence of decoyinine), and, finally, the influence of spoOA on the induction by purine starvation. The prg::lacZ fusions were localized on the genomic map. Except forprg-82::lacZ, which maps very close to the citB locus, none of the fusions were- mapped to loci previously reported to contain purine-regulated genes (27), and, therefore, they probably define new purine-responding genes. As shown in Table 5, the prg-82::1acZ fusion maps very close (>98% cotransduction) to the citB locus encoding the aconitase enzyme. A frequency of 99% cotransduction corresponds to a distance of less than 2 kb in our system. That prg-82::1acZ might be a citB allele is supported by three observations: (i) the two loci are in proximity, (ii) induction of both genes is independent of the spoOA gene product, and (iii) both genes are induced by decoyinine (6). According to reference 34, purine starvation of strain ED159 (purM) (blocked in the general pathway to IMP) results in low levels of both ATP and GTP, corresponding to a 20-fold reduction in the ATP pool and a 4-fold reduction in the GTP pool compared with the levels found in a wild-type strain growing in the absence of purine. Starvation of strain ED250 (purA), which is blocked in the adenine-specific pathway, results in very low levels of ATP (approximately 1% of the wild-type level) and an unchanged GTP pool. Guanine starvation of a strain defective in the guanine-specific pathway (guaA) leads to a 2-fold reduction and a 1.3-fold increase of the GTP and ATP pools, respectively. Identical changes in the purine nucleoside triphosphate pools have been observed during guanine starvation of a B. subtilis guaB mutant (31). On the basis of their responses to different kinds of purine starvation, we have divided the prg::lacZ fusions into two types (Fig. 5). One type (type 1) is induced by all three kinds of purine starvation (prg-71::1acZ, -74, and -76). The other type (type 2) (prg-26::1acZ, -42, -69, and -82) is induced only by specific starvation for ATP (in the purA strain) and by general starvation for ATP and GTP (in the purM strain) but not by specific starvation for GTP. The expression of all of the type 2 fusions resembles the pur operon expression, in that they are induced by general starvation as well as specific adenine starvation but not by specific starvation for guanine (34). prg-69::1acZ is the only fusion which is repressed by purine (5to 6-fold) in a wild-type strain. A 5- to 6-fold repression of the pur operon expression by hypoxanthine in a wild-type strain has previously been reported (34). This may indicate that prg-69::1acZ and the pur operon are regulated by a common purine-controlled mechanism.
282
SAXILD ET AL.
J. BACT1ERIOL.
0-
a)
0.
I-)
FIG. 5. Classification of the prg::lacZ fusions. Rectangles represent different types of responses to purine starvation. Type 1 fusions show induction during specific GTP starvation; type 2 fusions do not. Circles, different kinds of SpoOA-influenced gene expression; open boldface numbers, decoyinine-induced fusions.
The response of the prg::lacZ fusions to the presence of decoyinine was also investigated. Decoyinine blocks the synthesis of GTP by inhibiting the enzyme GMP synthetase, which converts XMP to GMP (Fig. 1). A 2-fold decrease in the GTP pool and a 1.2-fold increase in the ATP pool have been observed after 2 h of incubation of a wild-type B. subtilis strain with decoyinine (18). Hence, regarding changes in the purine nucleotide pools, guanine starvation caused by the addition of decoyinine is identical to that obtained by guanine starvation of a guaB or a guaA mutant (31, 34). In accordance with this, we found that all of the type 1 fusions induced by specific guanine starvation were also induced by addition of decoyinine. Furthermore, none of the type 2 fusions were induced by decoyinine, except prg-82::1acZ. Since the presence of guanine blocks induction of prg-82::lacZ by decoyinine, it is unlikely that decoyinine should affect gene expression by means other than through the cellular amount of guanine nucleotides. The dissimilarity in the induction patterns of prg-82::1acZ could therefore reflect differences in starvation conditions. The starvation conditions obtained by adding decoyinine resemble those found during starvation of a guaA mutant. Decoyinine inhibits the activity of GMP synthetase, while the guaA strain is GMP synthetase deficient. It has been reported that guanine starvation of a guaB mutant resulted in the accumulation of IMP, while XMP but not IMP accumulated in a guaA strain (33). Different effects on the levels of other intermediates in the biosynthesis pathways may also affect the induction pattern.
Treatment with decoyinine has been shown to induce massive sporulation in B. subtilis (24). It is also known that cultures
of leaky B. subtilis purine auxotrophs have a high titer of spores after cessation of batch growth in minimal medium, indicating that purine deprivation is sporogenic (10). Our findings that cultures of the leakypurL8 strain contain spores during growth in the absence of purine are in agreement with this observation. We therefore analyzed whether the expression of the prg::lacZ fusions was controlled by the spoOA gene product, which is a central regulatory component in the sporulation cascade in B. subtilis (9). A majority (five of seven) of the reported fusions show spoOA-influenced expression. However, no strict correlation between induction by decoyinine and the requirement for the spoOA gene product for normal prg::IacZ expression was found; spoOA-influenced expression was found
in all type 1 prg::lacZ fusions, but it was also found in prg-26::lacZ and -42, which do not respond to decoyinine. Although affected by SpoOA, the prg-26, -74, and -76 genes were not induced in Schaeffer's nutrient broth sporulation medium. Since nutrient broth most likely contains purine, it may indicate that these genes, besides the SpoOA-affected mechanism, are under the control of an additional system. The other system could be purine repression. Our data show that cultures of strain HH41, which have been grown for many generations in the absence of purine, consist of both growing and sporulating cells. One may therefore ask whether expression of the spoOA-associated prg::lacZ fusions is induced in all of the cells of the culture or exclusively in the sporulating subpopulation. The latter possibility seems not to be the case, because our results show that all of the prg::lacZ fusions are immediately induced after the exhaustion of purine in a purine-limited growth medium. At that point, we may expect all of the cells in the culture to be in the same growth state. The actual role of the spoOA gene product in prg::lacZ expression cannot be assessed from the present data. However, it has been shown that the SpoOA protein may function as an activator or a repressor of transcription (30, 38). In Fig. 5, we have classified the prg::lacZ fusions on the basis of their response to purine starvation and the presence of decoyinine and on the influence of the SpoOA protein on their expression patterns. When arranged in this manner, five classes appear. The gene regulation mechanisms responding to purine may be acting at the level of transcription initiation, transcription elongation, or, in the case of prg-42::lacZ, at the translational level. Several operons contain combinations of these regulatory mechanisms, i.e., the pur operon in B. subtilis (7). Even at a single step such as transcription initiation, several different regulatory proteins may be acting (i.e., deoP2 in E. coli [36]). The different responses of the lacZ fusions to the spoOA mutation and to starvation for ATP or GTP, respectively, are therefore not surprising. However, the number of isolatedprg::lacZ fusions probably does not represent the total number of prg genes. For example, no fusions to the promoters expressing the pur or gua genes, which are known to be induced by partial purine starvation, were isolated. Because of the well-known instability of certain B. subtilis genomic DNA fragments in high-copy-number plasmid vectors in E. coli, we do not expect that the pSGMU-based genome libraries represent all parts of the B. subtilis chromosome. We believe that as more fusions are characterized, the numbers in the different regulatory classes (Fig. 5) will increase. Finally, we may conclude that our data emphasize the importance of decreased levels of cellular purine nucleotide pools as a signal for sporulation induction in B. subtilis. Furthermore, partial purine starvation most likely represents a stress situation so serious that a number of genes, including sporulation-associated ones, are induced in order to guide the cell either to adaptation or sporulation. However, the mechanisms by which the concentrations of intracellular purine affect the prg::lacZ fusion expression still remain unsolved. Even though the collection of seven prg::lacZ fusions is far from a complete fusion library for purine-responding genes, it provides a very useful tool for isoiation of regulatory mutants involved in the purine stimulon. Experiments to pursue this goal have been initiated in our laboratory. Both cis- and trans-acting mutations are expected to be found. These studies should, it is hoped, result in identification of trans-acting components controlling several prg fusions and maybe also the pur operon.
PURINE-REGULATED GENES IN B. SUBTILIS
VOL. 176, 1994 ACKNOWLEDGMENTS We appreciate the help of Tove Atlung for carefully reading the manuscript and for critical comments. This work was supported by grant 16-4604.H from the Danish Technical Research Council and received financial support from the Lundbech Foundation, the Carlsberg Foundation, and the Saxild Family Foundation.
REFERENCES 1. Andersen, L., M. Kilstrup, and J. Neuhard. 1989. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of glnLG and purR genes in the regulation of codA expression. Arch. Microbiol. 152:115-118. 2. Atkinson, M. R., and S. H. Fisher. 1991. Identification of genes and gene products whose expression is activated during nitrogenlimited growth in Bacillus subtilis. J. Bacteriol. 173:23-27. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Boylan, S. A., M. D. Thomas, and C. W. Price. 1991. Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor orB of Bacillus subtilis. J. Bacteriol. 173:7856-7866. 5. Dedonder, R. A., J.-A. Lepesant, J. Lepesant-Kejzlarova, A. Billault, M. Steinmetz, and F. Kunst. 1977. Construction of a Kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl. Environ. Microbiol. 33:989-993. 6. Dingman, D. W., M. S. Rosenkrantz, and A. L. Sonenshein. 1987. Relationship between aconitase gene expression and sporulation in Bacillus subtilis. J. Bacteriol. 169:3068-3075. 7. Ebbole, D. J., and H. Zalkin. 1987. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for the de novo purine nucleotide synthesis. J. Biol. Chem. 262:82748287. 8. Errington, J. 1986. A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis. J. Gen. Microbiol. 132:2953-2966. 9. Errington, J. 1993. Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol. Rev. 57:1-33. 10. Freese, E., J. E. Heinze, and E. M. Galliers. 1979. Partial purine deprivation causes sporulation of Bacillus subtilis in the presence of excess ammonia, glucose and phosphate. J. Gen. Microbiol. 115:193-205. 11. Haldenwang, W. G., C. D. B. Banner, J. F. Ollington, R. Losick, J. A. Hoch, M. B. O'Conner, and A. L. Sonenshein. 1980. Mapping a cloned gene under sporulation control by insertion of a drug resistance marker into the Bacillus subtilis chromosome. J. Bacteriol. 142:90-98. 12. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. 13. He, B., A. Shiau, K. Y. Choi, H. Zalkin, and J. Smith. 1990. Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction. J. Bacteriol. 172:4555-4562. 14. He, B., J. M. Smith, and H. Zalkin. 1992. Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR. J. Bacteriol. 174:130-136. 15. Jaacks, K. J., J. Healy, R. Losick, and A. D. Grossman. 1989. Identification of genes controlled by the sporulation-regulatory gene spoOH in Bacillus subtilis. J. Bacteriol. 171:4121-4129. 16. Jensen, K. F., U. Houlberg, and P. Nygaard. 1979. Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyla-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity. Anal. Biochem. 98:254-263. 17. Lopez, J. M., A. Dromerick, and E. Freese. 1981. Response of guanosine 5'-triphosphate concentration to nutritional changes and its significanse for Bacillus subtilis sporulation. J. Bacteriol.
146:605-613. 18. Lopez, J. M., C. L. Marks, and E. Freese. 1979. The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis. Bio-
283
chem. Biophys. Acta 587:238-252. 19. Meng, L. M. 1989. Ph.D. thesis. University of Copenhagen, Copenhagen, Denmark. 20. Meng, L. M., M. Kilstrup, and P. Nygaard. 1990. Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli. Eur. J. Biochem. 187:373-379. 21. Meng, L. M., and P. Nygaard. 1990. Identification of hypoxanthine and guanine as the corepressors for the purine regulon genes of Escherichia coli Mol. Microbiol. 4:2187-2192. 22. Miller, J. H. (ed.). 1972. Experiments in molecular genetics, p. 135-139. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Miller, J. H. (ed.). 1972. Experiments in molecular genetics, p. 352-355. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Mitani, T., J. E. Heinze, and E. Freese. 1977. Induction of sporulation in Bacillus subtilis by decoyinine and hadacidine. Biochem. Biophys. Res. Commun. 77:1118-1125. 25. Mantsala, P., and H. Zalkin. 1992. Cloning and sequence of Bacillus subtilispurA and guaA, involved in the conversion of IMP to AMP and GMP. J. Bacteriol. 174:1883-1890. 26. M0lgaard, H. 1980. Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. J. Biol. Chem. 255:8216-8220. 27. Piggot, P. J., M. Amjad, J.-J. Wu, H. Sandoval, and J. Castro. 1990. Genetic and physical maps of Bacillus subtilis 168, p. 493-543. In C. R. Harwood and S. M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley and Sons Ltd., Chichester, England. 28. Price, V. A., I. M. Feavers, and A. Moir. 1989. Role of sigma H in expression of the fumarase gene (citG) in vegetative cells of Bacillus subtilis 168. J. Bacteriol. 171:5933-5939. 29. Rolfes, R. J., and H. Zalkin. 1988. Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. J. Biol. Chem. 263:19653-19661. 30. Satola, S. W., J. M. Baldus, and C. P. Moran, Jr. 1992. Binding of SpoOA stimulates spoIIG promoter activity in Bacillus subtilis. J. Bacteriol. 174:1448-1453. 31. Saxild, H. H., and P. Nygaard. Unpublished data. 32. Saxild, H. H., and P. Nygaard. 1987. Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs. J. Bacteriol. 169:2977-2983. 33. Saxild, H. H., and P. Nygaard. 1988. Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis. Mol. Gen. Genet. 211:160-167. 34. Saxild, H. H., and P. Nygaard. 1991. Regulation of levels of purine biosynthetic enzymes in Bacillus subtilis: effects of changing purine nucleotide pools. J. Gen. Microbiol. 137:2387-2394. 35. Schaeffer, P., J. Millet, and J.-P. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 54:704711. 36. S0gaard-Andersen, L., J. Martinussen, N. E. M0llegaard, S. R. Douthwaite, and P. Valentin-Hansen. 1990. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12. J. Bacteriol. 172: 5706-5713. 37. Steiert, J. G., R. J. Rolfes, H. Zalkin, and G. V. Stauffer. 1990. Regulation of the Escherichia coli glyA gene by the purR gene product. J. Bacteriol. 172:3799-3803. 38. Strauch, M., V. Webb, G. Spiegelman, and J. A. Hoch. 1990. The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene. Proc. Natl. Acad. Sci. USA 87:1801-1805. 39. Vandeyar, M. A., and S. A. Zahler. 1986. Chromosomal insertions of Tn917 in Bacillus subtilis. J. Bacteriol. 167:530-534. 40. Wilson, H. R., and C. L. Turnbough, Jr. 1990. Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12. J. Bacteriol. 172:3208-3213. 41. Wilson, R. L., L. T. Stauffer, and G. V. Stauffer. 1993. Roles of the GcvA and PurR proteins in negative regulation of the Escherichia coli glycine cleavage enzyme system. J. Bacteriol. 175:5129-5134.
