Vol. 140, No. 2

JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 671-679

0021-9193/79/11-0671/09$02.00/0

Regulatory Nucleotides Involved in the Rel Function of Bacillus subtilis TOYOKAZO NISHINO,t JONATHAN GALLANT,`* PETER SHALIT,' LINDA PALMER,1 AND TIMOTHY WEHR2 Department of Genetics, University of Washington, Seattle, Washington 98195,' and Varian Instrument Corporation, Walnut Creek, California 945982 Received for publication 29 August 1979

We have examined the accumulation of polyphosphorylated nucleotides in Bacillus subtilis in relation to the function of the rel gene. Our results are as follows. (i) During inhibition of isoleucine activation by O-methylthreonine, wildtype B. subtilis cells accumulate unusual nucleotides with the chromatographic and chemical properties of pppApp, ppApp, pppGpp, ppGpp, pGpp, and ppGp. (ii) During the carbon source downshift elicited by inhibiting glucose uptake, we observed accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iii) At the end of log phase in sporulation medium, we observed a small transient accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iv) We were unable to detect a nucleotide with chromatographic behavior expected for pppAppp under any conditions. (v) The rel mutant of Swanton and Edlin (Biochem. Biophys. Res. Commun. 46:583-588, 1972) did not accumulate any of these polyphosphorylated nucleotides under any of the conditions examined. (vi) The rel mutant is unimpaired in sporulation. We conclude that one or more of the nucleotides we have detected may be involved in controlling the specificity of transcription during the stringent response, but none of them are required for sporogenesis.

exponential growth to the stationary phase (1820). They have reported preliminary chemical characterization of three of the new nucleotides which suggest that they are ppApp, pppApp, and pppApp and have speculated that certain of these nucleotides may be involved in the control of sporogenesis (18-20). Murao et al. have discovered an enzyme in certain Streptomyces species hich producv a great variety of 3' pyrophophorylated nucleotides by transfer of the beta-gamma pyrophosphoryl group of ATP to appropriate acceptors (14-16). In B. subtilis, limited aminoacylation of tRNA triggers a response similar to that of the stringent control in E. coli (9, 21). Moreover, Swanton and Edlin have isolated a mutant, termed rel, which is seemingly analogous to relA of E. coli: in the mutant strain, blocked aminoacylation fails to elicit either accumulation of ppGpp and pppGpp or control of stable RNA synthesis (21). In view of the reports from Rhaese's laboratory, we were interested in determining whether unusual nucleotides other than ppGpp and pppGpp are involved in stringent control in B. subtilis. We have therefore examined the production of a variety of highly phosphorylated t Present address: College of Agriculture, University of nucleotides in an isogenic relI/rel pair of B. subtilis strains. Osaka Prefecture, Sakai 591, Japan.

In Escherichia coli, limited aminoacylation of tRNA elicits a regulatory response termed stringent control, which is characterized by greatly reduced rates of synthesis of the stable (rRNA and most tRNA) species, and numerous other adjustments in the pattern of transcription and in intermediary metabolism (3, 4, 8). Stringent control is defined as a unitary mechanism by single mutations in the reU gene which abolish the whole complex of adjustments. the prdidct of the reLA gene has been identified as a ribosome-bound pyrophosphotransferase which mediates the formation of two unusual nucleotides, pppGpp and ppGpp (1, 2, 12). A wealth of evidence, some of which remains controversial in detail, indicates that stringent control is mediated by diverse regulatory effects of these two nucleotides (3, 4, 8) and possibly of their derivatives (17). Other microorganisms may enjoy a metabolism of 3' phosphorylated nucleotides considerably more complicated than that of E. coli. Rhaese et al. have reported that Bacillus subtilis cells produce a variety of unusual polyphosphorylated nucleotides, including pppGpp, pppGpp and others, during the transition from

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MATERIALS AND METHODS The strains of B. subtilis were a lysine-requiring mutant of strain 168 (BR16 in our catalog) and its rel derivative (BR17 in our catalog). Both strains were obtained from Gordon Edlin. They were cultivated at 370C under forced aeration in a Tris-glucose medium containing 0.1 M Tris (pH 7.4), 0.1 mM KH2PO4, sodium citrate (0.42 mg/ml), MgSO4.7 H20 (0.21 mg/ ml), (NH4)2SO4 (1 mg/ml), FeCl3 (0.32 ,tg/ml), glucose (0.2%), and the following amino acids: lysine, proline, glycine, alanine, glutamic acid, aspartic acid, and arginine, all at 100 ,ug/ml; and cysteine, methionine, tyrosine, tryptophan, and phenylalanine, all at 40 Lg/ ml. Sporulation medium was the combination of nutrient medium (Difco) and inorganic salts described by Fortnagel and Freese (7). Growth was monitored tubidimetrically at 720 nm in a Beckman DB spectrophotometer; an absorbancy of 1.0 corresponds to approximately 1.2 x 109 cells. Nucleotides were labeled by the addition of [32P]phosphoric acid, at a few hundred microCuries per milliliter, at least one generation before extraction of samples. Small molecules were extracted by addition of formic acid to a final concentration of 0.1 N in the cold; after 30 min on ice, the extracts were adjusted to a pH of about 6 by the addition of one-quarter volume of 1.0 M Tris (pH 8.0) and centrifuged, and the supernatant was frozen for subsequent nucleotide analysis. Polyphosphorylated nucleotides were resolved by thin-laye; chromatography on PEI-cellulose in the following two-dimensional system: 4 M HCOOH + 1 M LiCl in the first dimension, followed by methanol

washing and development in 1.5 M KH2PO4 (unadjusted) in the second dimension. In some cases, the order of the two dimensions was reversed (see below and Fig. 1). With Brinkmann plates (but not others), this system affords excellent resolution of polyphosphorylated guanosine and adenosine nucleotides (Fig. 1A). In all separations, authentic markers (obtained from ICN or from P.L. Laboratories) were included and identified by UV absorption. Samples chromatographed were typically 20 p1 of cell extract. Data are reported as nanomoles of nucleotide per optical density unit of cells in culture. Nucleotide levels were quantitated by conventional methods of liquid scintillation spectrometry and corrected for background radioactivity (and trailing) by counting appropriate blank regions of chromatograms. The specific activity of the medium (which varied from 2 x 105 to 5 x 105 cpm/nmol of phosphate) was established by counting small portions together with each set of experimental samples, under identical counting conditions. The material we designate "MS3" was generally sufficiently well resolved from GTP (Fig. 1B) to permit its preparative isolation from the chromatogram. However, the separation system risks considerable artifactual hydrolysis of ppGpp to ppGp. This can be avoided by reversing the order of the solvents, as we have shown elsewhere (17); this method affords good resolution of MS3 (Fig. 1C) and was generally used to prepare the material for chemical studies. Typically, the material was isolated by scraping the appropriate region of several replicate chromatograms and transferring the scrapings to a small Quik-Sep column (Is-

4S.

IN 0 ops)AI)Pp ) l,l)Gpp

.

i pppA

4

0

A,o

I

O_

C/ .., .i-:-

A

:'I

'-C

FIG. 1. Resolution of3apyrophosphorylated nucleotides. (A) Positions of the authentic standard compounds, present at about 0.03 p.mol each; their positions were identified by UV absorption (under the shortwave lamp of a Chromato- Vue, Ultra- Violet Products, Inc.) and are marked by irregular solid circles. The same group of authentic standards was included in the experimental studies and are circled in the same way in Fig. 2 and 5. The 32P-labeled extract run together with the standards in (A) is from exponentially growing cells of strain BR16; heavy labeling ofpppG and pppA is evident. (B) Authentic standards were ppppA, pppG, pppA, and pGpp (reading up along the vertical dimension). The 32P-labeled extract run on the same chromatogram was prepared from a culture of BR16 subjected to O-methylthreonine inhibition. (C) The same standards and a similar 32P-labeled extract from O-methylthreonine-inhibited cells of BR16 were run together, with the order of the two solvents reversed, as described in the text. However, the autoradiogram is shown in the same orientation as (A) and (B), with the HCOOH-LiCl eluent (here the second dimension) still in the vertical direction, and the KH2PO4 eluent (here the first dimension) still in the horizontal direction.

Rel FUNCTION OF B. SUBTILIS

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nine led to the accumulation of nucleotides which comigrated with pppGpp, ppGpp, pppApp, and ppApp (Fig. 2A). In the rel mutant, no such accumulation occurred (Fig. 20). To confirm the identity of the labeled compounds, we isolated each of them by preparative thin-layer chromatography. The purified compounds were then tested for alkali lability, a characteristic feature of 3' pyrophosphorylated nucleotides, together with authentic standards. In the cases of pppGpp, ppGpp, pppApp, and ppApp, the labeled material was hydrolyzed at the same rate as the respective authentic standard and produced a degradation product which migrated identically in our two-dimensional sepRESULTS aration. These observations confirm the chemiStringent response of B. subtilis. In the cal identification of pppApp and ppApp in B. wild-type strain, inhibition of isoleucyl tRNA subtilis reported by Rhaese and Groscurth (20). synthetase by the addition of O-methylthreo- However these chemical structures should be

olab). The column was washed with methanol and then with 0.01 M Tris (pH 7.4) to remove the seconddimension, thin-layer chromatography eluent and assorted contaminants; the nucleotide material was then eluted with 2 M triethylammoniumbicarbonate, which was in turn removed by several cycles of evaporation. The isolated material was then dissolved in water for further analysis. For measurement of the proportion of heat-resistant prespore cells, culture portions were diluted in nutrient medium and plated with or without 10 min of incubation at 80°C. Cell counts, and the enumeration of phase-bright spore-forming cells, were done with a Zeiss phase-contrast microscope with x160 or x250 magnification.

A

IB

1--

.i

C

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FIG. 2. Polyphosphorylated nucleotides of B. subtilis. Strains BR16 (rer) and BR1 7 (rel) were labeled with 32p and polyphosphorylated nucleotides were analyzed as described in Materials and Methods. The authentic markers (whose positions are circled) are as in Fig. 1A. The panels are as follows: (A) BR16 30 min after addition of 2 mg of O-methylthreonine per ml; (B) BR16 in exponential growth; (C) BR1730 min after addition of 2 mg of O-methylthreonine per ml; (D) BR1 7 in exponential growth.

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regarded as tentative (see Discussion). Table 1 reports the kinetics of accumulation of these four nucleotides after O-methylthreonine inhibition. A low and barely significant basal level of all four nucleotides was observed in the rel mutant, but no increase was observed in the presence of O-methylthreonine. We should add that the measurements of ppApp are not very accurate because of trailing from the GTP spot; levels in the vicinity of 0.01 nmol per absorbancy unit are virtually semiquantitative. Nonetheless, the increase in this compound during the stringent response in the rel+ strain is clear (Fig. 2A), and material isolated from such chromatograms checked out as ppApp in rechromatography and the alkaline hydrolysis tests summarized above. Two more magic spots. Fig. 2A reveals the presence, in rel+ cells subjected to the stringent response, of a phosphorylated compound which migrates just above GTP. Better resolution of this material, which comigrates with the two isomers ppGp and pGpp, is shown in Fig. 1B and 1C. We have observed a compound of similar chromatographic properties in E. coli; we designate the compound MS3 and have shown that the E. coli MS3 is ppGp (17). Since neither ppGp nor its isomer pGpp have previously been reported in B. subtilis, we carried out more detailed characterization of the B. subtilis MS3, based on the methods we used to establish the structure of the E. coli compound (17). MS3 of B. subtilis is absorbed by Norit and labeled by [14C]guanosine, indicating that it is a purine nucleotide. It is resistant to periodate oxidation and does not complex borate ion, indicating the presence of a substituent on the 2' or 3' position (17). The nucleotide residue, obtained after alkaline phosphatase hydrolysis, proves to be unmodified guanosine (Fig. 3). Therefore, the substituent on the 2' or 3' position must be one or more of the phosphate groups. These properties are each consistent with

either isomeric structure ppGp or pGpp. The two isomers can be cleanly distinguished by their contrasting sensitivities to hydrolysis by alkali or by venom phosphodiesterase. Under our standard conditions, pGpp is completely hydrolyzed to the diphosphate product in less than 24 h of incubation in alkali and insensitive to 48 h of incubation in phosphodiesterase, whereas ppGp shows exactly the converse behavior (Table 2). Rather to our surprise, purified MS3 showed heterogeneous behavior in both tests: about 70% was stable to prolonged alkaline hydrolysis but sensitive to venom phosphodiesterase, whereas about 30% was sensitive to alkali but stable to prolonged phosphodiesterase treatment (Table 2). The fraction which was insensitive to phosphodiesterase proved to be alkali labile (Table 2). These observations suggest that B. subtilis MS3 in fact constitutes a mixture of the two isomers. Although we have been unable to resolve them satisfactorily in any thin-layer chromatographic system, good resolution can be obtained by high-pressure liquid chromatography; with this method, we have been able to resolve labeled MS3 into two distinct components which comigrate with ppGp and pGpp (Fig. 4). (The earlier peak of radioactivity in the chromatogram presumably reflects pGp formed by hydrolysis of pGpp during preparation and shipment to California for analysis.) We do not report levels of MS3 in Table 1 because the material was not fractionated into its two components. At its peak during the stringent response, MS3 reaches a level about 30% as high as that of ppGpp. Of this, at least 30% and probably somewhat more must be pGpp, judging by the chemical studies reported above; the lability of pGpp makes it difficult to estimate the quantity lost in preparation. Our minimal conclusion is that both ppGp and pGpp are formed during the stringent response in significant and

TABLE 1. Nucleotide levels in B. subtilis during the stringent responsea Nucleotide levelb BR16

Time (min)

BR17

pppGpp ppApp pppApp GTP ppGpp pppGpp ppApp pppApp 0.002 0.023 0.006 0.01