Vol. 127, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1047-1057 Copyright C 1976 American Society for Microbiology

Genetic Control of the glp System in Bacillus subtilis VIVEKA LINDGREN* AND LARS RUTBERG Department ofBacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden

Received for publication 23 March 1976

In pleiotropic negative glycerol utilization mutants (GlpP, mutants) of Bacillus subtilis, glycerol kinase and sn-glycerol 3-phosphate (G3P) dehydrogenase are noninducible. GlpP, mutants also fail to take up exogenous [14C]G3P. To study the regulation of the glp system in B. subtilis phenotypically, Glp+ revertants were isolated from GlpPI mutants. Four classes of revertants were identified: phenotypically, wild type; Rl type, which contains an informational suppressor, R2 type, which produces G3P dehydrogenase constitutively; and R3 type, with a temperature-sensitive Glp phenotype producing G3P dehydrogenase constitutively at permissive temperature (32°C). The properties of the revertants indicate that the glpP, locus codes for a protein with a positive regulatory function.

Bacillus subtilis can grow on glycerol as sole carbon source. The first two steps in the catabolism of glycerol in B. subtilis are mediated by a glycerol kinase (EC 2.7.1.30) and an sn-glycerol 3-phosphate (G3P; stereo-specific numbering according to Hirschmann [14]) dehydrogenase (EC 1.1.99.5) (20, 24, 29). In vitro, the activity of the G3P dehydrogenase, assayed in a crude lysate, is not stimulated by exogenous nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (24). Higher activity of G3P dehydrogenase is found in B. subtilis grown in a medium with glycerol or a racemic mixture of GlP plus G3P as main carbon source compared to growth in a medium with casein hydrolysate or an amino acid mixture as carbon source (20, 24, 29). The stimulating action of glycerol is dependent on its phosphorylation (20, 29). Also, the activity of glycerol kinase can be increased by addition of glycerol or GlP + G3P to the medium (20, 26, 29; see also reference 24). Mutants of B. subtilis specifically unable to grow on glycerol as sole carbon source (Glp mutants) have been isolated in various laboratories. Three classes have been found: GlpK mutants lacking glycerol kinase activity (20, 29), GlpD mutants lacking G3P dehydrogenase activity (20, 24, 25), and GlpP, mutants having a pleiotropic phenotype in that both glycerol kinase and G3P dehydrogenase are noninducible (20). The pleiotropic phenotype of GlpP1 mutants includes resistance to the antibiotic fosfomycin (formerly designated phosphonomycin; 13, 15, 20). A mutant with impaired glycerol permeation over the cell membrane has also been characterized (28, 29).

The glpK, glpD and glpP, loci are linked to each other and to the gtaC locus as assayed by transformation, the relative order being glpP, glpK glpD gtaC (20). In a previous report we suggested that glpP, represented a regulatory locus controlling the expression ofglpK and glpD (20). In this paper we will first present experiments to show that GlpP, mutants are also unable to take up externally added G3P, as suggested by their resistance to fosfomycin (13, 15, 20). The properties of a set of pleiotropic GlpP, mutants and different types of phenotypically Glp+ revertants of these mutants will then be described. On the basis of the results of these experiments, we will present a model for regulation of the glp system in B. subtilis. MATERIALS AND METHODS Bacterial strains. The bacterial strains used are listed in Table 1. For a comment on the Glp nomenclature, see reference 20. The pho mutation in BR95Pho reduces alkaline phosphatase activity assayed according to Le Hegerat and Anagnostopoulos (18) to less than 5% of that found in Pho+ bacteria. The pho mutation is linked to ilvC by PBS1-mediated transduction (data not shown). Media. Two kinds of minimal salt solution were used. One is the basal minimal salt solution described by Garen and Levinthal (8). This medium, supplemented with 0.3 mM inorganic phosphate, was used in [14C]G3P and [3H]glycerol transport assays and in some other experiments (indicated in the text) in which a low phosphate content was desirable. In all other cases the minimal salt solution was that described by Anagnostopoulos and Spizizen (1) supplemented with 10-5 M MnCl2. This minimal salt solution contains about 100 mM inorganic phosphate. Carbon source was added at a final

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TABLE 1. B. subtilis strains used Straina

W168 BR95 GtaC BR95Pho GlpD8-2

Relevant properties

Wild type trpC2, pheAl, ilvCl trpC2, lys-3, metB10, gtaC Lacks alkaline phosphatase activity Lacks NAD-independent G3P dehydrogenase ac-

Origin or derivation J. Spizizen J. Spizizen F. E. Young NTG, BR95 NTG, W168

tivityb GlpK21 GlpK11-4

Lacks glycerol kinase activity NTG, GlpP,18-R2 Lacks glycerol kinase activity NTG, W168 Noninducible glycerol kinase and G3P dehydroGlpP13-8 NTG, W168 genase activities; deficient G3P uptake NTG. W168 Noninducible glycerol kinase and G3P dehydroGlpP,6-5 genase activities; deficient G3P uptake Noninducible glycerol kinase and G3P dehydroNTG, W168 GlpP,6-9 genase activities; deficient G3P uptake GlpP,7-1 Noninducible glycerol kinase and G3P dehydroNTG, W168 genase activities; deficient G3P uptake GlpP,9-3 Noninducible glycerol kinase and G3P dehydroNTG, W168 genase activities; deficient G3P uptake Noninducible glycerol kinase and G3P dehydroGlpP112-6 NTG, W168 genase activities; deficient G3P uptake GlpP,18 Noninducible glycerol kinase and G3P dehydroNTG, W168 genase activities; deficient G3P uptake Wild type like Glp+ phenotype at 45°C; poor NTG, BR95GlpP,18 BR95GlpP,18-R1 growth in general at 32°C Constitutive G3P dehydrogenase activity; basal, Spontaneous Glp+ revertant GlpP,18-R2 noninducible glycerol kinase activity; deficient from GlpP,18 G3P uptake Constitutive G3P dehydrogenase activity at NTG, BR95GlpP,6-5 BR95GlpP,6-5-R3 32°C; parental GlpP, phenotype at 45°C Constitutive G3P dehydrogenase activity at NTG, BR95GlpP,6-9 BR95GlpP,6-9-R3 32°C; parental GlpP, phenotype at 45°C Constitutive G3P dehydrogenase activity at NTG, BR95GlpP,12-6 BR95GlpP,12-6-R3 32°C; parental GlpP, phenotype at 45°C a The variousglp mutations and also the pho mutation were introduced into other strains by congression. This is indicated in the text as BR95GIpP,18, GtaCGlpP,18, etc. b NAD, Nicotinamide adenine dinucleotide. concentration of 5/liter when not otherwise indi- cin and incubated for 20 h at 370C. Colonies of differcated. When glycerol was used as sole carbon source, ent sizes were picked at random, purified, and then citrate was omitted from the minimal salt solution. tested for growth on glucose and glycerol. Only Min-CH plates are minimal glucose plates supple- strains that did grow on glucose and glycerol were mented with 0.5 g of casein hydrolysate (Difco) per further investigated. Spontaneous Phm' mutants liter. Fosfomycin sensitivity was assayed on plates were isolated from overnight cultures of BR95 grown with the LP medium described by Miki et al. (23) but in basal minimal medium supplemented with rewith sodium lactate omitted. quired amino acids and with glycerol as carbon Both the minimal medium and the LP medium source. The cultures were diluted 1:10 and then were solidified by the addition of 18 g of Noble agar spread on LP plates containing 0.1 mM fosfomycin. (Difco) per liter. After 20 h of incubation, colonies were picked, puriThe strains were maintained on tryptose blood fied, and tested for growth on glucose and glycerol. agar base (TBAB; Difco) plates. A mutant lacking alkaline phosphatase activity Mutant isolation. GlpP, mutants were isolated was isolated from an NTG-mutagenized culture of from B. subtilis W168 after treatment with N- BR95 by the filter disk procedure described by Miki methyl-N'-nitro-N-nitrosoguanidine (NTG; K & K et al. (23). The phosphatase mutation was then inLaboratories, Inc., Hollywood, Calif.) as described troduced by congression into Glp and Phmr strains previously (20). to reduce possible hydrolysis of the substrate in Mutants with reduced G3P transport activity are ['4C]G3P transport experiments. Congression is a selected as fosfomycin-resistant types, Phmr (13, 15, procedure used to introduce markers, which cannot 20). Phmr strains were isolated both after NTG be directly selected, into new strains. Deoxyribonutreatment and as spontaneous mutants. Appropri- cleic acid from bacteria carrying such a marker is ate dilutions of NTG-mutagenized W168 cultures used at saturating concentration to transform anwere spread on LP plates containing 1 mM fosfomy- other strain for a marker that can be easily selected.

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The resulting transformants are then scored for the presence of the unselected marker. As before (20), all the mutants described in the text were isolated 120 independently. All of the glpP, mutations, and also the NTG-induced phmr mutations, were subsequently introduced into strain BR95 by congression. 100 Phenotypic Glp+ revertants were isolated from GlpP, mutants either as spontaneous revertants or after NTG or ethyl methane sulfonate (Sigma > __80 Chemical Co.) treatment. For isolation of spontaneous Glp+ revertants, the GlpP, strains were grown NE overnight in PB (antibiotic medium 3; Difco) or in a x 60 C minimal medium with casein hydrolysate (5 g/liter) E c 0 as carbon source and then washed with minimal salt E X solution and spread on minimal glycerol plates, 2 which were incubated for 60 to 90 h at 32, 37, and .2_450C. Colonies appearing on the minimal glycerol * 0 20 plates were picked and restreaked for isolation of a E single colonies. Revertants strains isolated at 32°C tn _ were then tested for growth on minimal glycerol O _ 80 plates at 45°C and vice versa. The procedure for 0 60 20 40 NTG mutagenesis was the same as described previInduction time (minutes) ously (20), except that NTG was used at a concentration of 20 ,ug/ml. The frequency of Glp+ revertants B was found to be higher at the lower NTG concentration. Ethyl methane sulfonate treatment was car120[ried out according to Meynell and Meynell (22). The phenotypes of all of the Glp+ revertants obtained after ethyl methane sulfonate mutagenesis were 100 either wild type or R3 (see below) and were not used in further experiments. When not otherwise indicated, the selection procedure after NTG and ethyl 80 methane sulfonate mutagenesis was the same as for selection of spontaneous revertants. Cultivation of bacteria. Cultivation of bacteria 60 _ for preparation of cell extracts and for uptake assays was as described previously (20), with the exception ` E 40 that the induction period was extended to 90 min when the bacteria were grown at 32°C. In experi- a Ex ments in which the growth temperature was 37 or Z 45°C, the induction period was 60 min. The growth -L 20 rate in the minimal casein hydrolysate medium U c used in induction experiments is similar for strain BR95 and its mutant derivatives. Growth of mu100 80 60 0 40 20 tants lacking G3P dehydrogenase activity is slowed Induction timne (minutes) down by addition of glycerol or GlP + G3P. Figure 1 FIG. 1. Induction of glycerol kinase (A) and G3P shows the induction of glycerol kinase and G3P dehydrogenase in strain BR95 grown at 32 and 37°C. dehydrogenase (a) by 40 mM GIP + G3P in strain Chloramphenicol prevents the GlP + G3P-induced BR95 grown at 37°C (A) and at 320C (B). The bacteincrease of glycerol kinase and G3P dehydrogenase ria were grown and induced as previously described activities (data not shown). The cells were in expo- (20). The only difference between control cultures nential growth throughout the induction period. We (open symbols) and induced cultures (closed symdo not know the reason for the decline after a brief bols) was the presence of GIP + G3P in the latter. period of rapid enzyme synthesis; it may be due to as with a racemic mixture of GlP + G3P (data not catabolite repression or inducer depletion. Preparation of extracts for enzymatic assays. shown). Contamination of the GlP + G3P mixture Extracts for assays of glycerol kinase and GP dehy- with glycerol was found to be about 3% determined drogenase activities were prepared as described pre- according to Freedberg and Lin (7). Thus, glycerol phosphate dehydrogenase in B. subtilis is active viously (20). Assays of glycerol-catabolizing enzymes. The ac- with G3P but not with GlP as substrate, as is the tivities of glycerol kinase and G3P dehydrogenase case inEscherichia coli (19). The enzyme is therefore were assayed as described by Lin et al. (19). All designated as G3P dehydrogenase. The protein conenzyme assays were performed at 25°C. Specific ac- centrations of the extracts were measured according tivity ofthe dehydrogenase assayed in a crude lysate to Lowry et al. (21) with bovine serum albumin with G3P as substrate is two to three times as high (Sigma Chemical Co.) as standard. -

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Assays of uptake of [14C]G3P and [3lHlglycerol. The experimental protocol is a modification of that of Hayashi et al. (12). The bacteria used in the uptake experiments all contained the pho mutation from BR95Pho. Cells were centrifuged and washed twice with basal minimal medium at room temperature (20 to 25°C) and then suspended to approximately 108 cells/ml in basal minimal medium containing 50 ,ug of chloramphenicol per ml. [U-14C1G3P (128 ,tCi/,mol; final concentration, 2 ,uM) or [1(3)3H]glycerol (180 ,uCi/,mol; final concentration, 2 ,tM) was added to 10 ml of the bacterial suspension, which was continuously aerated at room temperature. One-milliliter samples were withdrawn at various intervals, immediately collected on membrane filters (type HA, Millipore Corp.), and washed with 20 ml of basal minimal medium within 25 s. The filters were soaked in 0.1 M phosphate buffer for 20 h before use. After collection of samples, the filters were dried, put into scintillation vials with 10 ml of scintillation liquid {10 g of 2,5-diphenyloxazole and 0.125 g of 1,4-bis-[2-(5-phenyloxazolyl)]-benzene per liter toluene}, and counted in a Nuclear-Chicago Mark I liquid scintillation computer, model 6860. Efficiency of counting was 20% for [3H]glycerol and higher than 95% for [14C]G3P. A blank sample value obtained by filtering 1 ml of medium with labeled substrate, but without bacteria, was subtracted from each uptake value. The viable cell concentration of the bacterial suspension was determined by plating appropriate dilutions on TBAB plates 10 min after addition of the radioactive substrate. No change of viable cell concentration during the experimental period (35 min) was found (data not shown). Transformation and PBS1 transduction experiments. The procedures for transformation and transduction were as described in a previous report (20). Scoring of recombinants. Glp+ transformants or transductants were picked with sterile toothpicks to minimal glycerol plates and then, to score the GtaC phenotype, to two TBAB plates, one of which was spread with approximately 108 429 phage particles. These plates were incubated overnight at 32°C. GtaC mutants lack phosphoglucomutase activity and are unable to glucosylate their teichoic acid. They are resistant to phage 429 (30). To score fosfomycin sensitivity, the recombinants were picked to LP plates with the appropriate concentration of fosfomycin and incubated for 24 h. Chemicals. Crystalline rabbit muscle G3P dehydrogenase, adenosine 5'-triphosphate, nicotinamide adenine dinucleotide, 3-(4,5-dimethyl thiazolyl-2)2,5-diphenyltetrazolium bromide, phenazin methosulfate, and DL-a-GP (i.e., a racemic mixture of GlP + G3P) were purchased from Sigma Chemical Co., St. Louis, Mo. Hydrazine was obtained from Eastman Kodak Co., Rochester, N.Y.; glycerol was from BDH Chemicals Ltd., Poole, England; [1,33H]glycerol was from The Radiochemical Centre Ltd., Amersham, England; and [U-14C]G3P was from New England Nuclear Chemicals GmbH, Dreieichenhain, West Germany. The disodium salt of fosfomycin was a gift from Merck, Sharpe & Dohme International.

J. BACTERIOL.

RESULTS Uptake of G3P. Experiments to measure uptake of G3P in B. subtilis are complicated by the presence of polyglycerol teichoic acid in the membrane and cell wall. In our uptake experiments with [14C]G3P, about 25% of the radioactivity taken up during 35 min is recovered in a wall membrane fraction. We have performed experiments, however, to measure uptake of G3P in wild-type bacteria and various Glp mutants. Our main purpose with these experiments was to compare G3P uptake in GlpP, and Phmr mutants, respectively, with uptake in wild-type bacteria and GlpK and GlpD mutants. The results of these experiments allow us to make some qualitative conclusions regarding G3P uptake in B. subtilis. G3P is taken up by B. subtilis wild-type bacteria and GlpD and GlpK mutants. Figure 2A shows uptake in a GlpD mutant. In wild-type bacteria and in the GlpD mutant the rate of accumulation of radioactivity from [14C]G3P is increased by inducer during middle or late exponential growth phase, whereas growth in the presence of inducer does not affect the rate of uptake in bacteria in early exponential growth (data not shown). GlpK mutants accumulate radioactivity from [14C]G3P at a slower rate than GlpD or wild-type bacteria and inducer does not affect this rate. Addition of a 1,000-fold excess of unlabeled G3P to GlpD bacteria in an uptake experiment leads to displacement of about 75% of the accumulated radioactivity (data not shown). Accumulation of [14C]G3P in a GlpD mutant is inhibited by 10-3 M 2,4-dinitrophenol (data not shown). GlpP1 mutants are pleiotropic, they show low noninducible levels of glycerol kinase and G3P dehydrogenase, and they can also grow at concentrations of the antibiotic fosfomycin not tolerated by wild-type bacteria, GlpK mutants, or GlpD mutants (20). In E. coli and other bacteria, fosfomycin can enter the cells via the G3P transport system (13, 15). All GlpP1 mutants isolated (see Table 1) show strongly reduced uptake of [14C]G3P (Fig. 2B). We have isolated fosfomycin-resistant mutants that grow on glycerol (Phmr mutants). These mutants fail to take up [14C]G3P (data not shown). We can thus conclude from the above experiments that the GlpPI mutants, besides being noninducible for glycerol kinase and G3P dehydrogenase, are also defective in G3P uptake. Glycerol has been shown to enter B. subtilis by facilitated diffusion (28). We have found that

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crosses place the mutations on the opposite side of glpK from gtaC (Table 3). They are closely linked to previously described glpP1 mutations by transformation (Table 4). Mutations glpP138 and glpP,9-3 show no detectable recombination, and the other pair, glpP16-9 and glpP1126, gives less than 0.1% recombination in transformation (Table 4). They may be mutations at the same site, although they have been isolated from independently mutagenized cultures. Since GlpP, mutants are pleiotropic and have been obtained by NTG mutagenesis, it is important to show that they do not carry additional mutations that contribute to the observed phenotype (11) and that the mutations do not affect the metabolism of other carbon sources. All of our GlpPj mutants grow as well as wildtype bacteria on the following substances as sole carbon source: glucose, sucrose, fructose, mannitol, sorbitol, and casein hydrolysate; they can also take up [3HIglycerol. Thus, they differ from the Ctr mutants described by Gay et al. (9). Next, GIpP118 deoxyribonucleic acid was used to transform BR95 to Phe+, and the transformants were purified and tested for the presence ofthe glp marker. Of 21 Glp transformants tested, all were resistant to fosfomycin and had low, noninducible levels of glycerol kinase and G3P dehydrogenase. The GlpP, phenotype thus TABLE 2. Activities of glycerol-catabolizing enzymes in wild type and in GlpP, mutantsa

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30 40 Time (minutes) FIG. 2. Uptake of [14C]G3P in strains BR95GlpD82Pho (A) and BR95GlpP,18Pho (B). The bacteria were grown and induced as previously described (20), with the following inducers: 4 mM GIP + G3P (0), 40 mM GIP + G3P (-), or 20 mM glycerol (A). Control cultures (A) did not receive any inducer; otherwise the composition of the medium was the same as for the induced cultures. 0

radioactivity from exogenous [3H]glycerol is accumulated by wild-type bacteria and GlpD and GlpPI mutants, but not by GlpK mutants. Isolation of GlpP1 mutants. Four new independent pleiotropic Glp mutants were isolated as described previously (20; Table 2). The mutations are linked to the gtaC locus by transformation, and three-factor transformation

W168 W168 BR95 BR95 BR95

GlpP,3-8 GlpP,3-8 BR95GlpP,3-8 BR95GlpP,6-5 BR95GlpP,6-5 BR95GlpP,6-5 GlpP,6-9 GlpP,6-9 BR95GlpP,6-9 GlpP,7-1 BR95GlpP,7-1 BR95GlpP,7-1

Inducee -

40 hnM Glp + G3P -

40 mM G1P + G3P 20 mM glycerol -

40 mM GlP + G3P 20 mM glycerol -

40 mM G1P + G3P 20 mM glycerol -

40 mM GlP + G3P 20 mM glycerol -

40 mM GIP + G3P 20 mM glycerol

Glycerol G3P dehykinase drogenase 38

Genetic control of the glp system in Bacillus subtilis.

Vol. 127, No. 3 Printed in U.S.A. JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1047-1057 Copyright C 1976 American Society for Microbiology Genetic Contr...
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