THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 13, Issue of July 10, pp. 6866-6875, 1981 Printed in U. S.A.

Initiation of Bacillus subtilis Sporulation by the Stringent Response to Partial Amino Acid Deprivation*

(Received for publication, November 1, 1980, and in revised form, February 24, 1981)

Kozo Ochi, Jagan C. KandalaS, and Ernst Freese From the Laboratory of Molecular Biology, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20205

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 m~ L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial “strin- gent response” occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (mlA) mutants, the changes of guanine nucleo- tides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.

Sporulation of Bacillus subtilis usually starts when the bacilli are in a medium that contains only slowly metabolizable carbon or nitrogen sources, or no phosphate. In contrast, cells do not sporulate in a medium containing excess glucose, ammonium ions, and phosphate. When in earlier experiments auxotrophic mutants, grown in such a glucose medium plus the required supplements, were suddenly deprived of such a supplement, leaky purine auxotrophs sporulated, whereas all our amino acid-, pyrimidine-, or vitamin-requiring mutants did not (1-4). Nonleaky purine mutants could also sporulate if a purine precursor was supplied at concentrations allowing growth only at a reduced rate. The results demonstrated that a partial, but not a complete, deprivation of purine nucleo- tides, in particular guanine nucleotides, initiates sporulation (4). Furthermore, nucleotide measurements showed that the concentrations of GDP and GTP decreased under all sporu- lation conditions, whereas the concentrations of the other nucleotides increased under some and decreased under other sporulation conditions (5).

Mutants able to sporulate in the presence of glucose have also been isolated in Bacillus cereus (6) and Bacillus mega- terium ( 7 ) . Although the more efficiently sporulating strains were purine auxotrophs, several amino acid auxotrophs could also sporulate to some extent. As the importance of residual substrate synthesis for sporulation was not recognized at the time, the leakiness (or residual growth rate) of these mutants in the absence of the known growth requirement was not

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department of Chemistry, University of South Carolina, Columbia, SC 29208.

examined. Because in B. subtilis purine auxotrophs sporulated after complete purine removal only if they were leaky, the examined amino acid auxotrophs might not have sporulated after amino acid removal simply because they were not leaky. Therefore, we wanted to determine whether partial limitation of an amino acid would initiate sporulation. For this purpose we needed conditions under which the concentration in the medium of an amino acid, or a metabolic precursor of it, would determine the intracellular amino acid concentration; differ- ent degrees of this limitation would be revealed by different growth rates. Such conditions are usually difficult to attain in simple auxotrophs because most amino acids and some of their precursors are actively transported into cells, with K, values in the micromolar range. We used three approaches to prevent or circumvent this problem. One approach was to use mutants that were not only auxotrophic but also deficient in the active transport of the required supplement; they required high concentrations of this compound for normal growth. Another approach was to compete the uptake of one amino acid by another amino acid taken up by the same transport system. The third approach was to use a D-amino acid which was utilized much more slowly than the corresponding L- amino acid. In all three cases, we found that partial limitation of amino acid supply caused a rapid decrease of GTP and initiated sporulation.

Amino acid limitation also produces the “stringent re- sponse” whereby the rate of RNA (especially rRNA) synthesis and of numerous other cellular reactions, including GTP synthesis, are severely reduced. This effect is accompanied by the increase in the intracellular concentrations of ppGpp’ and pppGpp, compounds which may be responsible for the strin- gent response (8,9). The stringent response can be avoided by the introduction of a relaxed (rel) mutation, which allows continued RNA synthesis upon amino acid deprivation and which prevents the accumulation of ppGpp and pppGpp. A reEA mutation has been isolated in B. subtilis by Swanton and Edlin (10); Smith et al. (11) have mapped it and identified it as relA by its inability to produce the “stringent factor,” needed for the synthesis of ppGpp and pppGpp. To analyze the effect of the stringent response on sporulation, we have isolated mutants, isogenic with the above auxotrophic and transport-deficient strains, which contain in addition a relA mutation. We show that partial limitation of the amino acid supply caused a rapid increase of ppGpp and pppGpp, a corresponding decrease of GTP, and extensive sporulation in the stringent but not the relaxed strains. However, the relaxed strains could sporulate well, even under conditions of partial amino acid limitation, if the concentration of GTP was suffi- ciently decreased by other means (than the stringent re-

’ The abbreviations used are: ppGpp, guanosine-5’-diphosphate-3’- diphosphate; pppGpp, guanosine-5‘- triphosphate - 3” diphosphate; Omv, DL-a-oxo-P-methyl-n-derate.

6866

Stringent Response Initiates B. subtilis Sporulation 6867

sponse); for example, by addition of decoyinine, which did not cause an increase in the concentrations of ppGpp and pppGpp.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Their Isolation-The strains of B. subtilis used are listed in Table I. Transformation was performed as previ- ously described (12). To identify strains defective in active transport of the oxo-acid precursors of Ile and Val (gene symbol kauA), we picked colonies which grew especially slowly on plates with synthetic medium containing low concentrations (0.1 mM) of D L - ~ - o x o - ~ - methyl-n-valerate (oxomethylvalerate) and Val. To select stringent (rel') mutants, after transforming this trait into the rel- recipient 61885, we used a variation of the method described to US by J. Gallant.' We plated the cells on synthetic medium containing 0.005% (w/v) casamino acids (to initiate growth) and 200 pg/ml of each D-L- norleucine, DL-serine hydroxamate, and S-aminoethyl-L-cysteine. Stringent strains produced slightly larger colonies. Stringent mutant 61884 was derived from the relaxed strain 61883 as described above. The presence of the relaxed or stringent trait was also determined by measuring the rate of incorporation of [3H]uracil (45 pM, 0.1 p c i / d ) into the trichloroacetic acid (5% final concentration)-precipitable portion of amino acid-starved cultures by a method similar to Swan- ton and Edlin (10). About one-third of the large colonies tested showed the stringent response.

Growth and Sporulation Conditions-Synthetic medium con- tained 10 mM ammonium sulfate, 5 mM potassium phosphate (pH 7.0), 100 mM morpholinopropanesulfonate (adjusted to pH 7.0 with KOH), 2 m~ MgC12, 0.7 mM CaC12, 50 pM MnC12, 5 ~ L M FeCb, 2 pm thiamine, 55 m~ D-glucose, and 20 mM L-glutamate (adjusted to pH 7.0 with KOH). Auxotrophic requirements were satisfied by the addition of 0.25 m~ L-tryptophan and other compounds as described in the text. Nutrient sporulation medium contained 0.8% nutrient broth (w/v, Difco), 1 m~ MgC12, 0.7 mM CaCh, 50 pM MnCL, 10 pM FeC13, 10 m~ potassium phosphate buffer (pH 7.0), 5 mM potassium acetate (pH 7.0), and supplements as required by auxotrophs.

Strains were inoculated from frozen cultures onto plates containing tryptose blood agar base (3.3% w/v; Difco), plus 10 mM aspartate for strains 61883 and 61884), and incubated for 6 to 8 h at 37 "C. The cells were collected into synthetic medium and inoculated at low ODm (0.0005 to 0.002) into flasks containing (%o volume 00 synthetic medium plus excess of any required compounds (0.25 m~ L-trypto- phan, 10 mM L-aspartate, 1 mM L-isoleucine, 2 mM DL-valine, 0.14 mM L-methionine, or 3.4 mM D-methionine). These "precultures" were shaken overnight at 37 "C. When the ODm of each culture reached 0.5, the cells were collected by fdtration through membrane filters (Nalge 125-ml filter unit, 50-mm diameter, 0.45 pm pore size, Nalge Co.), immediately washed with 10 to 20 ml synthetic medium lacking the supplement to be starved for, and suspended in this medium; 10- ml aliquots were distributed into flasks containing various amounts of the supplement and incubated at 37 "C with vigorous shaking. Growth was monitored by the ODm. Later, usually 10 h after cell transfer, the viable cell titer was measured by diluting the culture in 0.1 M potassium phosphate buffer (pH 6.5) plus 1 m~ MgClz and plating on tryptose blood agar base plates. The spore titer was measured by heating the diluted cuftures for 20 rnin at 75 "C and then plating.

Determination of Nucleotide Pools by High Pressure Liquid Chra- matogruphy-Cells were grown in 100-ml synthetic medium plus excess supplements to ODW = 0.3 to 0.5, collected on a membrane filter (100 mm diameter, 0.45 pm pore size, Schleicher and Schuell), immediately washed with 30 ml of synthetic medium lacking the supplement to be starved for, and the filters with cells were quickly transferred to 1-liter flasks containing 100 ml of the stated medium. After incubation for the indicated time, the cells of one flask at one time were rapidly collected as just described, and the filter was then laid upside down onto 1.5 ml of ice-cold formic acid (0.5 M) in a plastic Petri dish; the entire collection procedure took 7 s. The OD, was determined just before fdtration. After 60 min incubation at 4 "C, the membrane filter was removed by centrifugation (5,000 x g, 5 min), and the remaining cells were removed from the extract by a second centrifugation (5,000 X g, 10 rnin). The supernatant was filtered though a syringe (5 ml) fitted with a Millipore filter (Swinnex, 0.45- P Pore size, 13 mm diameter) to remove all remaining debris and then vacuum-evaporated (Speed Vac Concentrator, Savant Instru- ments) for 7 to 8 h. The residue was dissolved in 130 p1 of deionized

I_

J . Gallant, personal communication.

water. For the assay of nucleoside di- to pentaphosphates, 60 pl of the &solved residue were applied to a column of Partisil P x s 10/25 SAX [Whatman], SO,'- form, which had been washed with 0.1 N HzS04 and for 5 min with 7 m~ KHzPO4 adjusted to pH 4.0 by HaPOs. The column was part of a high performance liquid chromatography system (ALTEX model 420; UV detector, Schoeffel Inst. CO. Mon- ochrometer Model GM770). The nucleotides were eluted at a flow rate of 1.5 ml/min by a gradient made up of a low ionic strength buffer (7 mM KH2P04, adjusted to pH 4.0 by HaP04) and a high ionic strength buffer (0.5 M KH2P04 + 0.5 M Na&04, adjusted to pH 5.0 by KOH). The percentage of buffer with high ionic strength was in- creased for 20 min from 0 to 20%, for another 20 min from 20 to 47% for 5 min from 47 to loo%, and then remained for 20 min at 100%. By comparison with the peak area of standards, the amount of each nucleotide was determined. To normalize the picomoles of nucleotides found per ml of culture to the picomoles per amount of cells, the values were divided by the ODm values measured at the time of cell filtration; i.e. all values were expressed in picomoles per AMm (1 AMm or AM unit is the amount of cells which would produce an ODm = 1 if suspended in 1 ml).

Determination of Highly Phosphorylated Nucleotides by 3zPL In- corporation-Strains (61885 and 61886) were cultivated from a very low inoculum in synthetic medium containing potassium phosphate (2 mM), L-isoleucine (1 mM), and DL-valine (2 mM). When the ODm of the culture reached 0.05, 32P04 was added to give a specific activity of 50 Ci/mol of Pi. To label nucleotides completely, each culture was shaken until the ODW reached 0.5. The cells were harvested by filtration (Nalge 125-ml filter unit), rapidly washed with 10 ml of synthetic medium, containing DL-valine (0.6 mM) and DL-oxomethyl- valerate (0.4 m ~ ) , suspended in the same medium containing "'PO4 (2 mM, 50 Ci/mol of P,), and shaken at 37 "C. At the indicated times, I-ml aliquots were removed and filtered through membrane filters (pore size 0.45 pm, diameter 25 mm) which were then laid upside down on 0.1 ml of ice-cold formic acid (0.5 M) in a plastic Petri dish. After 1 h at 4 "C, the filter was removed, the formic acid extract was centrifuged (5,000 X g for 5 rnin), and 10 pl of the supernatant was applied to a point close to one corner and 2 cm from the edges of a thin layer plate (20 X 20 cm; Polygram CEL 300 PEI of Macherey- Nagel Co., Duren, West Germany). The plate had been washed previously and all anions had been exchanged by P,, as described (5) . Ten pg each of standard nucleotides were applied to the same point. The plate was first developed with 0.6 M LiCl, dried, washed for 5 min with methanol, and again dried. Then the plate was sprayed with 0.1 M potassium phosphate buffer, pH 7.5, to which urea (7 M final concentration) had been added. It was electrophoresed in the second dimension in the electrophoresis apparatus of the Inert Control Co., Woodburg, NY, containing Varsol as Coolant. The current was kept between 50 and 70 mA (using about 1500 V) for about 60 min. After complete drying, the plates were placed against X-Omat R X-ray film (Kodak Co.) and a light-intensifying screen (Cronex Xtra Life, Light- ning Plus, Dupont Co.) and incubated for 2 days at -70 "C. The radioactive spots corresponding to ppGpp and pppGpp were cut out and counted in 10 ml of Aquasol (New England Nuclear Co.). For every culture, the counts per min per pmol of phosphate were deter- mined using the phosphate concentration of samples measured ac- cording to the method of Ames and Dubin (13).

Chemical Co., St. Louis, MO, ppCpp and pppGpp from ICN Co., 32P, Chemicals-We purchased DL-oxomethylvalerate from Sigma

and [5,6-3H]uracil from New England Nuclear Co. Decoyinine was a gift of Dr. G . W. Whitfield of the Upjohn Co., Kalamazoo, MI. Other reagents came from commercial sources and were of analytical grade.

RESULTS

Isolation and Growth Properties of Mutants-We have isolated three types of auxotrophic mutants (61885 = ilvB kauA relA, 61883 = aspB reZA, 61950 = metC relA) as described under "Experimental Procedures." They all con- tained the relA mutation and are listed in Table I; irrelevant auxotrophic requirements (trpC) will be left out in the text. For the first two mutants, the corresponding stringent bel+) strains (61886 = ilvB kauA, 61884 = aspB) were isolated by transformation with rel+ DNA. These rel-/rel+ pairs are therefore isogenic. For the metC rel' auxotroph we used our standard strain (60015 = netC). In all rel+ strains, we ascer- tained the stringent response following amino acid starvation

6868 Stringent Response Initiates B. subtilis Sporulation

TABLE I Strains of B. subtilis used in this study

Strains Genotype Source

60005 Prototroph 60015 metC7 trpC2 our standard

61566 aspB66 aspTl trpC2 Tf a of 61565 by 61504 61848 ilvB-dell kauAl trpC2 CU965 of S. Zahler 61852 lys relA trpC2 from Swanton and Edlin 61879 ilvB-dell relA trpC2 Tf of 61852 by 61848 61881 ilvB-dell k a d l Tf of 61848 by 60005 61883 aspB66 relA trpC2 Tf of 61852 by 61566 61884 aspB66 trpC2 Tf of 61883 by 60015 61885 ilvB-dell kauAl relA Tf of 61879 by 61881 61886 ilvB-dell kauA1 Tf of 61885 by 60015 61950 metC7 relA trpC2 Tf of 61852 by 60015

Tf = transformation of the first strain by the second one.

= SB26 of E. Nester

by the low rate of uracil incorporation into acid-precipitable material (RNA), in contrast to the continued incorporation in the relaxed strains. The rel+ isoleucine- and valine-requiring auxotroph (61886 = ilu kau) grew at different rates when the medium was supplied with 0.6 mM DL-Valine and different concentrations of isoleucine precursor DL-oxomethylvalerate (Fig. lA) because it lacked active transport of oxomethylval- erate. Fig. 1B shows that the growth rate of the rel+ and rei- strains at a given oxomethylvalerate concentration were es- sentially the same. The aspartate auxotroph (61884 = aspB) grew at different rates when different concentrations of potas- sium aspartate were supplied because the 20 mM glutamate in the medium competed with the aspartate transport (Fig. IC). The relaxed asp auxotroph (61883) grew on limiting aspartate concentration slower than corresponding stringent strain (61884) although the strains were isogenic (Fig. 1D). In the re1 mutants, more aspartate was apparently drained into purine and pyrimidine nucleotides, which were used for the overproduction of RNA because addition of adenine and uracil (0.5 mM each) abolished the difference between the stringent and the relaxed strains almost completely (Fig. 1D). When cells of the methionine auxotroph (60015 = metC) were grown with L-methionine in the medium, they could subsequently not grow on D-methionine. But they adapted overnight to grow with D-methionine (3.4 m) and then grew at different rates if the medium contained different concentrations of D- methionine (Fig. 1E). The re1 strain grew at the same rates as the rel’ strain (Fig. IF) . Inside the cells, the D-methionine was converted to L-methionine by an inducible methionine racemase (see ~upplement).~

Sporulation under Conditions of Partial Amino Acid Dep- riuation-The ability of cells to sporulate upon amino acid starvation was demonstrated in the following manner. Cells were grown overnight from a very low inoculum in synthetic medium (which contained excess glucose, ammonium ions, and phosphate) plus the required supplements at concentra- tions exceeding those needed to optimally satisfy the auxo- trophic requirements (see legend to Fig. 1 ) . During exponen- tial growth (ODm = 0.5), the cells were washed on membrane filters and transferred to fresh medium containing different concentrations of the compound limiting the growth rate. Fig. 2 shows that the three stringent (reZ+) mutants sporulated

Portions of this paper (including Figs. SI to S5, Tables SI and SII, and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20014. Request Document 80 “2360, cite author(s), and include a check or money order for $6.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

(mM)

5 1.5

1 0.4 0.3

0.2

0.1

B s 2 1

0

0.5

“ = o w 0 hours

hours

ImM)

10 E 3.4

6.7 1.3

5 0.67

B 8 2

0.042

0.014

1 0

0.5

0’40 2 4 6 8 10

hours

61886 bel+)

.- E I-

.- c“ l o o

8 6 0 n

4o0 0.4 0.8 1.2 1.6 2.0 A

DL-oxomethvhtalerate (mM)

L-aspartate (mM)

“1, 0.01 011 1 I 10

D-methionine (mM)

FIG. 1. Growth of auxotrophs at various concentrations of a required nutrient. A + B, strains 61885 and 61886 were grown in synthetic medium (see “Experimental Procedures”) containing 2 mM DL-valine and 1 rn L-isoleucine. At ODW = 0.5, the cells were washed on membrane filters with 10 ml of synthetic medium lacking the supplement and suspended in synthetic medium containing 0.6 mM DL-valine and different concentrations of oxomethylvalerate. C + D, strains 61883 and 61884 were grown in synthetic medium containing 10 mM Asp. At ODW = 0.5, the cells were washed and suspended in synthetic medium plus different concentrations of Asp. E + F, strains 60015 and 61950 were grown in synthetic medium containing 3.4 mM D-methionine. At ODW = 0.5, the cells were washed and suspended in synthetic medium plus different concentrations of D-methionine. The growth curves in panels A , C, and E are for the stringent (rei+) strains. Numbers on the right of each curve represent the final concentration (millimolar) of the varied substrate. The doubling times (in panels B, D, and F) were determined from the growth curves between 1 and 3 h after transfer. Symbols used for B, D, F: stringent M, relaxed 0--0. For the relaxed asp strain (61883) cells were also transferred to synthetic medium containing 0.5 m~ each Ade and Ura in addition to limiting concentrations of Asp. The doubling times are marked by a”€!.

Stringent Response Initiates B. subtilis Sporulation 6869

poorly both with excess or with no growth supplement; but they sporulated extensively at an intermediate concentration of the supplement. The optimal spore titer was 1 to 3 X lo' spores/ml, only slightly less than the titer observed when sporulation was initiated by specific guanine deprivation (4). In contrast, the three relaxed (reM) mutants did not sporulate at any concentration of the growth-limiting compound (Fig. 2). These mutants were nevertheless capable of sporulating in nutrient sporulation medium, where they produced 4 to 5 X 10' spores/ml) . Their sporulation could also be induced by the addition of decoyinine (2 m ~ ) regardless of whether they were grown with excess amino acid or whether conditions of partial amino acid deprivation were used that allowed optimal spor- ulation of the stringent strains (Fig. 3, see also supplement Table

Nucleotide Changes under Optimal Sporulation Condi- tions-We measured the changes of intracellular nucleotide concentrations following the cell transfer to the growth-lim- iting substrate concentrations that allowed optimal sporula- tion conditions. (For nucleotide concentrations observed after cell transfer to different substrate concentrations, see supple- ment Fig. S5). As described under "Experimental Proce- dures," we extracted the cells with 0.5 M formic acid just before the cell transfer and at different times thereafter. Usually, we measured the nucleotides by high pressure liquid chromatography; as this method was not sensitive enough for accurate determination of the highly phosphorylated guanine nucleotides in the relaxed strains, we labeled the nucleotides in some experiments by growing the cells in the presence of 32P04 for two or more generations before the step-down and continued with the same specific activity of 32P04 after the step-down. As Fig. 4 shows, following the cell transfer to

10 r lv/

10

I I I 1 0.5 1 1.5 2

DL- ImM) 1IYr A

4 f : ; ~ m l r : l l I_ > lffo"o.ol 0.05 0.1 0.5 1 5 10

DmMinina ImMl

FIG. 2. Induction of sporulation after limitation of the amino acid requirement in stringent and relaxed strains. Strains were grown and transferred to media containing growth-limiting concen- trations of the required supplements as in Fig. 1. Spore titers were measured 10 h after transfer. Symbols used O"+ for stringent strains and o"-o for relaxed strains.

Reloxed

0 ' 10 il il 40 50 60'7120 1

Ml"

FIG. 3. Time course of sporulation and of changes in GTP pools in ilv kau strains 61886 (rel+) and 61886 (rel-). Both strains were cultivated and transferred to the new medium containing the limiting amount of oxomethylvalerate (0.4 m ~ ) that produced optimal sporulation in the reZ+ strain as determined in Fig. 2; the re1 strain 61885 was also transferred to the limiting oxomethylvalerate medium containing decoyinine (2 m). GTP was measured in formic acid extracts, and heat-resistant spore titers were determined as described under "Experimental Procedures." Symbols used M, 61886 (stringent); C " O , 61885 (relaxed); A, 61885 (relaxed) with decoyinine. The vertical bars represent the values of two experiments or the standard deviation of three or four experiments.

growth limiting conditions both ppGpp and pppGpp tran- siently increased in the stringent strain whereas they increased only little in the relaxed mutants (at most to 10% of the level observed in the stringent strains). In the stringent strains, the rapid increase in ppGpp and pppGpp correlated with a rapid decrease in GTP. In the relaxed strains, GTP decreased much more slowly, reaching 47% (strain 61885), 40% (strain 61950), or 30% (strain 61883) of the initial intracellular GTP concen- trations after 1 h. As Fig. 2 shows, this decrease did not suffice to initiate sporulation. Addition of decoyinine (2 mM) to the relaxed strains produced, after some delay, a greater decrease of GTP than did amino acid deprivation alone (shown for the ilv mutant in Fig. 3) without increasing ppGpp and pppGpp (not shown); decoyinine addition caused extensive sporulation in all three relaxed strains.

Sporulation during Long Term Growth under Conditions of Oxomethylvalerate Limitation, i.e. at a Reduced Growth Rate-In the stringent strain, following the initial effect of amino acid starvation, the concentrations of the highly phos- phorylated guanine nucleotides decreased again to a value only slightly higher than the initial one, and the GTP concen- tration increased again, eventually reaching a value similar to that of the relaxed strain. When the new balance between the reduced amino acid supply and the rates of RNA and protein synthesis is eventually established, most tRNA presumably is charged again so that much less ppGpp is then made and GTP can again be synthesized at a rate sufficient to allow growth at a reduced rate. The cells should then be able to grow slowly without much sporulation. If that is so, the transfer of cells to limiting amino acid concentrations, tran- siently reducing the GTP concentration to a very low value, should cause only a transient increase in sporulation. To check that possibility, we grew cells of the ilv kau mutant (61886) in synthetic medium, containing excess isoleucine and valine,

6870 Stringent Response Initiates B. subtilis Sporulation

OMV Starvation

61886 (red’)

u OO 20 40 60 80 100 120

min

Methionine Starvation

Min 200 m

- E f 150 150

480 80

8 a % 100 100

S

+ .-

z 60 60

40 40

61885 (ml) 20 20

OO 20 40 60 80 100 120 0 20 40 60 80 100120 OO 20 40 60 80 100120 0

min min min

Aspartate Starvation 180 r

OLl-~” u 0 20 40 60 80 100120 OO 20 40 60 80 100120

min min

GTP ATP UTP CTP FIG. 4. Changes in the intracellular concentrations of nu- 61886

cleoside triphosphates, ppGpp and pppGpp after cell transfer (stringent) to media partially deficient in amino acids and allowing max- 61885 imal sporulation. Strains, grown in Fig. 1, were washed and trans- (relaxed) ferred to new media allowing optimal sporulation of the reZ+ strains; 61884 i.e. containing 0.4 mM DL-oxomethylvalerate and 0.6 mM DL-valine (stringent) for 61885 and 61886,67 p~ D-methionine for strains 60015 and 61950, 61883 1.5 mM L-aspartate for strains 61883 and 61884. Cell extracts were (relaxed) prepared and evaluated by high pressure chromatography or for 60015 ppGpp and pppGpp in the ilu strains (61885 and 61886) by 32P-labeling (stringent) and subsequent thin layer chromatography and electrophoresis as 61950 described under “Experimental Procedures.” Just before the transfer (relaxed) of cells to conditions of amino acid deprivation (see inserts) the concentrations of nucleotides, measured in two to six separate exper- Symbols used are for GTP, M, ATP, o ” 0 , UTP, A-A; iments and averaged, were (in picomoles per AMw f S.D.). CTP, A-4 PPGPP; H , PPPGPP, w.

432 f 84 1273 f 122 559 f 57 263 f 55

404 f 69 1779 f 106 680 f 48 319 f 62

410 f 140 1217 f 174 504 f 27 218 f 22

448 f 31 1077 f 51 542 f 0 152 f 42

541 f 10 1620 f 33 788 f 2 439 f 19

376 f 10 1046 f 63 505 f 6 218 f 24

and transferred them at ODm = 0.3 to medium containing excess valine and 0.35 mM DL-oxomethylvalerate, the concen- tration allowing nearly optimal sporulation. Whenever the culture reached twice the original ODm, we diluted it 2-fold in the same medium. Therefore, the ODsoo as well as the oxomethylvalerate concentration were kept essentially con- stant for 40 h. (The doubling times remained at 90 min throughout the 40-h period.) At the time of dilution, the spore titer was measured. Following the initial cell transfer, the culture sporulated after about 6 to 8 h (see Fig. 5), but cells

growing at this time no longer sporulated later. This is shown by the fact that the spore titer decreased due to the dilution effect instead of remaining high as it would have done if each subculture could have sporulated with the same probability. The intracellular concentration of GTP, measured for the first 2 h at different times and then each time just before culture dilution, decreased rapidly in the first 5 min and then in- creased to 60% of the original level within 2 h; thereafter, it remained constant. The concentration of the other three nucleoside triphosphates changed initially as in Fig. 4, and

Stringent Response Initiates B. subtilis Sporulation 687 1

r

0 5 10 15 20 25 30 35 40 5001 hours

400 t

I I I , I 1 0 5 10 15 M 25 30 35 40

rloufs

FIG. 5. Changes in spore titer and GTP pool during long term culture of the stringent strain 61886 under condition of partial oxomethylvalerate starvation. Cells were grown in syn- thetic medium containing 1 m~ L-isoleucine and 2 mM DL-valine, to OD- = 0.3, washed and transferred to DL-oxomethylvalerate (0.35 mM) limiting medium; they were repeatedly diluted in this medium as described in the text. The spore titer was measured just before each dilution. To determine the GTP pool, cells were transferred to an initial culture in a flask containing 10 ml of oxomethylvalerate limiting medium in a I-liter flask. Whenever the ODw of the diluted culture in the main experiment had doubled, the volume of the culture was doubled by addition of the same growth-limiting medium. When- ever the volume exceeded 100 ml, 100 ml of culture were fiitered, the cellular nucleotides were extracted and measured as described under “Experimental Procedures.” Symbols used are: e, heat-resistant spores/&, A, GTP.

after 2 h, remained constant at the level of the zero time value.

These results showed that the cells continued to grow in the limiting oxomethylvalerate medium at the same reduced rate throughout the 40 h experiment but they eventually did not sporulate at a significant frequency. Nevertheless, these cells were still able to sporulate upon addition of decoyinine (2 mM) (see supplement, Table S2). To eliminate the possi- bility that the cells which continued to grow had mutated to sporulation deficiency, a portion of the last subculture was diluted and plated at 40 h, and 18 colonies were isolated. Cells of these isolates were grown in synthetic medium containing excess valine and isoleucine, and each was again exposed to the oxomethylvalerate downshift, they all sporulated, produc- ing spore titers (0.3 to 2 X lo7 spores/ml) similar to the original strain.

DISCUSSION

Our results demonstrate that amino acid auxotrophs which exhibit the stringent response upon amino acid starvation can sporulate when the amino acid supply is reduced but not completely blocked. To control the intracellular concentration of the amino acids, required by three different auxotrophs, via the extracellular concentration of the supplied compound we used three different methods, a transport mutation (kau) to reduce the rate of oxomethylvalerate uptake, competition by Glu to limit the rate of Asp uptake, and D-methiOnine instead of the actively transported L-methionine. When we transferred the cells to a medium containing the supplement at a concen- tration that allowed optimal sporulation, the concentrations of ppGpp and pppGpp rapidly increased and later decreased

again to a value only slightly higher than the original one (Fig. 4). Simultaneously, the concentration of GTP decreased rap- idly and later increased again slowly. (When no amino acid supplement was added these changes were even more drastic.) The corresponding relaxed strains did not sporulate at any amino acid concentration. In these cells, the concentrations of ppGpp and pppGpp increased only slightly and that of GTP decreased only slowly and to a limited degree (Figs. 3 and 4). This shows that the stringent response was required for spor- ulation resulting from amino acid deprivation. However, when the decrease of GTP in the relaxed strain was enhanced by the addition of decoyinine, which inhibits the synthesis of guanine nucleotides, the relaxed strain also sporulated. Fur- thermore, both the relaxed and the stringent strains growing in the presence of excess supplements sporulated better after decoyinine addition than during optimal amino acid depriva- tion (see supplement Table S2); the decoyinine addition caused a decrease of GTP without a concomitant increase of ppGpp and pppGpp. The nucleotide change common to all sporulation conditions is therefore the decrease of GTP rather than the increase of ppGpp and pppGpp. This agrees with earlier findings that all media conditions initiating sporulation also cause a decrease of GTP (and GDP), whereas the other nucleoside triphosphates increase in some and decrease in other cases (5). The increase of ppGpp observed under spor- ulation conditions due to partial amino acid deprivation may be responsible for the decrease of GTP because Gallant et al. (14) have found in cell extracts of Escherichia coli that IMP dehydrogenase, the f i t enzyme in the specific GMP pathway, is inhibited by ppGpp. The inhibition of IMP dehydrogenase during the stringent response of B. subtilis has also been demonstrated in uiuo.4 Although these results make it likely that the decrease of GTP alone is responsible for the initiation of sporulation, we have not excluded, by the experiments presented here, the possibility that the increase of ppGpp or pppGpp is more important than the decrease of GTP for the initiation of sporulation by the stringent response.

The optimal sporulation, observed at some intermediate concentration of the amino acid supplement, results from two opposing effects. On one hand, the concentration of the com- pound suppressing sporulation (GTP or a compound con- trolled by it) has to decrease enough to retard or prevent growth and to allow the initiation of sporulation. On the other hand, protein synthesis is required for the sporulation process. Therefore, a too severe amino acid starvation, although it might allow the initiation of sporulation, does not permit enough synthesis of proteins needed at critical times for the sporulation development. Presumably, the partial amino acid limitation allowing sporulation reduces protein synthesis more than does the direct partial GTP limitation (caused by decoy- inine) which can also initiate sporulation. Therefore, it is understandable that cells do not sporulate as efficiently after partial amino acid deprivation as after the more specific deprivation of guanine nucleotides in the presence of all other nutrients.

After the initial shock of amino acid deprivation, rel’ cells adapt to the presence of a reduced supply of an amino acid. This is indicated by the fact that the concentrations of ppGpp and pppGpp eventually decrease again, leveling off at a some- what higher concentration than that observed without amino acid deprivation (Fig. 4 ) . Similarly, the concentration of GTP eventually reaches a value that is only slightly lower than that without amino acid deprivation. Presumably, the rapid protein synthesis, which continues until the existing mRNA has been used up (9, 15), uncharges the tRNA that corresponds to the

J. M. Lopez, A. Dromerick, and E. Freese (1981) J. Bacteriol., in press.

6872 Stringent Response Initiates B. subtilis Sporulation

removed amino acid. When the rate of protein synthesis has Spores VZZ (Chambliss, G., and Vary, J. C., eds) pp. 277-285, slowed down, this tRNA can again be s&cientli charged by the now slowly supplied amino acid so that only a mild stringent response remains, resulting in a reduced rate of RNA and protein synthesis and thus of cell growth. The sporulation observed after cell transfer to partial amino acid deprivation is also largely due to this initial shock. When the cells grow for a long time under the conditions of partial amino acid deprivation, they maintain a constant slow growth rate; but after the initial burst of sporulation, they eventually sporulate at an only slightly higher frequency than when they grow rapidly in the presence of excess supplement (Fig. 5). These results demonstrate that differentiation of B. subtilis is initi- ated by quantitative changes in intracellular metabolites.

Acknowledgments-We thank Enid Galliers and Stuart Critton for their skilled assistance in some of the experiments.

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420-429

Stringent Response Initiates B. subtilis Sporulation

ISOUTION AND CHARACTERIZILTION OF STRlNGEPRl AND RELAXED STRAINS

4k 2 2t % min

6873

10 6 8

0 2 3

0 7

0 1

06 0 2 4 6 8 1 0

houn

6874 Stringent Response Initiates B. subtilis Sporulation

Stringent Response Initiates B. subtilis Sporulation

REFERENCES

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20. Hochscadt-Ozer. J., and Cashel, M . (19121 J. Biol. Chsm. x, 7067-7072.

0.1

2 0.06

0