Proteins That Interact with GTP during Sporulation of Bacillus subtilis

Vol. 171, No. 6

JOURNAL OF BACTERIOLOGY, June 1989, p. 2915-2918

0021-9193/89/062915-04$02.00/0 Copyright ©3 1989, American Society for Microbiology

Proteins That Interact with GTP during Sporulation of Bacillus subtilis C. MITCHELL AND J. C. VARY* Department of Biochemistry, University of Illinois at Chicago, Chicago, Illinois 60612 Received 10 November 1988/Accepted 21 February 1989

During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link [a-32P]GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either [0-32P]GTP or [_y-32P]GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming that GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms.

Sporulation of bacilli is generally thought to be a response to nutrient deprivation in the environment. Although the process of sporulation has been characterized morphologically, biochemically, and genetically, the molecular mechanisms of triggering sporulation and of spore germination are poorly understood. Clearly, important signal transduction pathways must be involved in these processes. For instance, guanine nucleotides have been implicated by Freese and co-workers to play an integral role in sporulation of bacilli (7, 11). A decrease in intracellular GTP concentration was associated with the early stages of sporulation in Bacillus subtilis, and sporulation could be stimulated either by the addition of decoyinine, an inhibitor of GMP synthetase, or by guanine starvation of a guanine auxotroph. Exactly where GTP exerts its effect is still speculative, as reviewed by Sonenshein (17). Other mechanisms of signal transduction could involve protein methylation, as recently reported by Bernlohr et al. (3), and changes in protein phosphorylation (M. Nikolopoulou, C. Mitchell, and J. C. Vary, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, 1107, p. 182). Guanine nucleotide-binding proteins could be involved in sensing nutrient deprivation as a signal for sporulation. In eucaryotes, GTP-binding proteins participate in many biological signal transduction processes such as hormonal regulation of adenylate cyclase, phototransduction in retinal rod photoreceptor cells, and the olfactory response in the olfactory epithelium (for reviews, see references 8, 12, and 18). In yeasts, a GTP-binding protein called SCG1, which has sequence homology with the above-mentioned proteins, was implicated in the pheromone response pathway (5) and therefore indirectly with sporulation. Also in yeasts, a pair of smaller GTP-binding proteins called RAS1 and RAS2 have some sequence homology to the signal-transducing GTPbinding proteins, especially at the GTP-binding site (19). By analogy to eucaryotic systems, it seems logical to think of GTP as playing a role in sporulation via GTP-binding proteins; however, GTP could also modify proteins by phosphorylation or guanylation. The results presented here show that all of the above occur during sporulation.

*

MATERIALS AND METHODS

Materials. [ao-32P]GTP and [-y-32P]GTP (600 Ci/mmol) were obtained from ICN Radiochemicals. Nitrocellulose paper (pore size, 0.1 ,lm) was obtained from Vangard International Inc. Chemicals for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories. B. subtilis DB100 (metB hisH) was strain NIG 1121, originally from Y. Sadaie (14) and obtained from R. Doi. B. megaterium QM B1551 (ATCC 12872) was obtained from H. S. Levinson. All other chemicals were reagent grade or better. Labeling cell proteins. Cells were grown in supplemented nutrient broth (15). Cell samples were collected by centrifugation at 5,000 x g, and the pellet was suspended at 37°C for 10 min in 4 ml of 0.1 M N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES) buffer (pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride and lysozyme (0.5 mg/ ml). The samples were then treated for 15 to 45 s at 2°C with a Branson W185 D sonic oscillator (21), except for samples of spores that required more sonication (13). The supernatant fractions were obtained after centrifugation at 10,000 x g for 10 min, and the soluble-protein concentration was determined (16). The procedure used to covalently label quanine nucleotide-binding proteins was exactly that used to label ras p21 (2) by cross-linking [a-32P]GTP to proteins with UV light. In a total volume of 40 ,ul, 100 to 200 ,ug of protein was incubated with a mixture containing 20 mM Tris (pH 7.8), 1 mM dithiothreitol, 100 mM NaCl, 2 mM MnCl2, 0.1 mg of bovine serum albumin per ml, 0.5 ,uM [at-32P]GTP (600 mCi/,umol), and 10 ,uM ATP to reduce GTP binding to ATP-binding proteins. The mixture was incubated at 0°C for 15 min and then illuminated with UV light (Mineralight lamp model R-52G UVP, Inc.); placed 5 cm above the samples for 20 min; 10 pul of 4 mM GTP and 25 ,ul of 0.18 M Tris (pH 6.8) containing 3% (wt/vol) SDS, 0.45 M dithiothreitol, 30% sucrose, and 0.003% bromphenol blue were then added. Samples were boiled for 3 min and loaded onto a 10% polyacrylamide gel, and electrophoresis was carried out by the method of Laemmli (10). To reduce background radioactivity, the proteins were electrophoretically blotted to nitrocellulose paper (0.3 A for 6 h) in a solution containing 19.2 mM Tris hydrochloride, 192.2 mM glycine, 0.015%

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FIG. 2. Comparison of B. subtilis proteins. Samples of cell growth were incubated with 0.5 p.M [a-32P]GTP or [y-32P]GTP (600 mCi/,Lmol) plus (lanes +) or minus (lanes -) cross-linking with UV light and compared as described in the legend to Fig. 1.

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FIG. 1. Proteins labeled with [a-32P]GTP by cross-linking with UV light. B. subtilis (a) or B. megaterium (b) were grown to mid-exponential (vegetative, lanes v) and early stationary (lanes to) phases of growth and mature spores (lanes s). The cells were disrupted, and equal amounts of protein (ca. 200 p.g) were incubated with 0.5 ,uM [a-32P]GTP and cross-linked with UV light; this was followed by electrophoresis, electrophoretic blotting, and autoradiography as described in the text. The letters indicate individual bands in B. subtilis, and the numbers refer to molecular mass standards (in kilodaltons).

SDS, and 20% methanol; the paper was then exposed to Kodak XAR-5 film with an intensifying screen. RESULTS AND DISCUSSION To investigate the presence of guanine nucleotide-binding proteins, we first studied samples of B. subtilis DB100 from three different stages of sporogenesis: vegetative growth, to (which represents both the end of exponential growth and the beginning of sporulation), and mature spores. The samples were broken by lysis and sonication, and the soluble proteins were incubated with [(x-32P]GTP at 0°C by the method of Basu and Modak (2) as described above. After the nucleotides were cross-linked to proteins with UV light, the proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose paper, and exposed to film (Fig. la). Several controls were also used and are discussed below. Fifteen major bands of radioactivity were observed; they were labeled A through 0. Vegetative cells (Fig. la, lane v) had 11 bands, A, B, D, E, G, H, K, L, M, N, and 0. By changing film exposure times, it was apparent that band E was probably two bands, but we did not resolve them; however, band K showed no evidence of multiple species (data not shown). Cells from stage to of growth had four additional bands (C, F, I, and J in lane to). Finally, mature spores contained nine radioactively labeled bands, which were in the approximate location of bands A, B, C, D, E, F, H, K, and N (lane s); the radioactivity above band A in lane s was in the stacking gel and was not present in other experiments. Identical results were found with B. subtilis 168 trp (strain L-4) from J. Hagemen, as well as two collections of B. subtilis sporulation mutants, obtained from the Bacillus Genetic Stock Center, Ohio State University, and from R. Doi, that represented mutations in all sporula-

tion stage loci from spoO to spoV. In other words, sample extracts from any of the mutants that were grown to stage to looked like samples of strain DB100 at to, whereas samples at later times from mutants blocked late in sporulation looked like strain DB100 late in sporulation with respect to the pattern of labeling with [a-32P]GTP. We also labeled cell proteins with two analogs of GTP, [-y-35SIGTP and [a-32P]P3-(4-azidoanilido)-Pl-5'-GTP, but we could not reduce the high backgrounds on either the gels or electroblots to identify individual bands as in Fig. 1. To determine whether these results were unique to B. subtilis, we performed similar experiments with B. megaterium. A distribution of radioactive bands was also observed that changed during sporulation (Fig. lb). If one looks only at the major bands for the purpose of comparison, bands at 72 and 63 kilodaltons (kDa) were present in all cell types. Four bands, with molecular masses of 57, 52, 48, and 46 kDa, were present in vegetative and stage to cells, and bands at 50 and 76 kDa were seen in spores only. We have tentatively identified the 76-, 72-, 57-, 52-, 48-, and 46-kDa bands as nucleotide-binding proteins on the basis of the criteria described below, whereas the bands at 63 and 50 kDa could be phosphorylated and/or guanylated. Several of these proteins corresponded in apparent molecular masses to proteins labeled in B. subtilis, but further analysis is required to prove whether they are the same proteins. Some insight into the nature of radioactive labeling can be obtained by considering the following. First, if GTP interacted with specific proteins by only noncovalent binding, those proteins would not be labeled without UV crosslinking of [a-32P]GTP. To test this, a B. subtilis cell extract corresponding to stage to of sporulation was incubated with [a-32P]GTP with or without UV light (Fig. 2). Of the 15 bands, 11 required cross-linking by UV light, as expected for a noncovalent GTP-binding protein, whereas bands C, H, K, and L did not, suggesting that these four bands became radioactive by some covalent modification and not just by binding of the nucleotide to a protein. As noted in Materials and Methods, all samples were diluted into the sample buffer of Laemmli (10), which contained 2% SDS, and they were analyzed on denaturing gels, which should cause the complete dissociation of any nucleotides bound to proteins by

VOL. 171, 1989

PROTEIN-GTP INTERACTIONS IN B. SUBTILIS

TABLE 1. Interactions of GTP with B. subtilis proteins Interaction of protein with': Band

A B C D E F G H I J K L M N 0

[a-32P]GTP

[y-32PJGTP

+UV

-UV

+UV

-UV

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+

+ + -

+ + -

+

-

-

-

+

-

-

-

+

-

-

-

+ + +

+ -

+

-

-

+

+

(kDa)b

(ka 83 74 72 66 61 57 52 45 40 38 36 32 28 25 22

Deduced interaction with GTP3"

B B

G, P B, P B B B G, P B B G G, P B B B

a The results shown in Fig. 2 are summarized as bands being present (+) or absent (-) on the gels. b The relative molecular masses of the proteins are based on their mobilities relative to standards. c Possible protein interactions with GTP are listed as noncovalent binding (B), guanylation (G), or phosphorylation (P), based on the radioactive labeling data.

noncovalent interactions. Second, the radioactivity in all bands was progressively reduced by competition with increasing concentrations of unlabeled GTP between 4 and 400 ,uM. Also, the addition of 2% SDS to the extracts and/or boiling them before the addition of [a-32P]GTP eliminated radioactivity in all of the protein bands both with and without UV treatment. Third, proteins with the same mobilities as bands E, F, and G were ADP-ribosylated with pertussis toxin (D. Rossignol, personal communication), as would be expected for certain eucaryotic GTP-binding proteins (8). Fourth, we examined the labeling of proteins with [.y-32P]GTP (Fig. 2) and found that bands C, D, H, and L were radioactive. None required UV cross-linking, and they presumably were phosphorylated by GTP. The above results are summarized in Table 1. By comparing the differential labeling, bands A, B, D, E, F, G, I, J, M, N, and 0 appear to be guanine nucleotide-binding proteins, since UV light was required to label them with [a-32P]GTP and they were not labeled with [_y-32P]GTP. In addition to binding [a-32P]GTP, band D (or another protein with similar mobility) also was labeled with [.y-32P]GTP, presumably by phosphorylation. Bands C, H, K, and L did not require UV cross-linking to be labeled with [a-32P]GTP, suggesting a protein modification such as guanylation, and three of these (bands C, H, and L or other proteins of similar mobilities) were also phosphorylated by [.y-32P]GTP. Further analysis of the isolated proteins will determine the exact chemical nature of these protein modifications. The above data show the following. First, B. subtilis and B. megaterium contain several apparent guanine nucleotidebinding proteins. Second, the amounts of the individual proteins change during sporulation. Third, sporulation-negative mutants were not lacking in any of these proteins by our assays. If any of these proteins could be involved in sensing GTP levels in the cell, such a protein would presumably be present in vegetative cells. In addition to known proteins such as translation initiation and elongation factors, these are, to our knowledge, the first data to show directly that bacilli have many guanine nucleotide-binding proteins.

2917

Recently, a protein of 52 kDa with no known function was reported on the basis of DNA sequence similarity between the spoOB2 gene and a GTP-binding consensus sequence (K. Trach and J. Hoch, Abstr. 10th Int. Spore Conf., p. 80, 1988). Also, another guanine nucleotide-binding protein, ERA, has been reported to be present in Escherichia coli, and it has no known function yet (1). Fourth, on the basis of the comparison of differential labeling summarized in Table 1, certain proteins were apparently modified with GTP by phosphorylation or guanylation. To our knowledge, guanylation of proteins would be considered rare, and we hope to pursue that interesting possibility. That certain proteins were phosphorylated agrees with our previous report that several proteins become phosphorylated during sporulation (Nikolopoulou et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1986) and with the fact that B. subtilis protein kinases can use ATP or GTP as a phosphate donor (C. Mitchell and J. C. Vary, unpublished results). Protein kinases in E. coli have also been reported to use either ATP or GTP (6). Some of the phosphorylated proteins could also be involved with chemotaxis as in E. coli (9); this could be tested in B. subtilis by using available chemotaxis mutants (20). Another possibility would be phosphorylation of isocitrate dehydrogenase or enzymes involved in sugar phosphotransferase systems as reviewed by Cozzone (4). These possibilities are being investigated. Our data provide another approach for investigating how GTP levels might be involved in signaling sporulation. In addition to changes in guanine nucleotide-binding proteins during sporulation, we observed what appears to be guanylation and phosphorylation by GTP. These types of protein modifications are most probably regulatory and could be important during sporulation. ACKNOWLEDGMENTS We thank M. Rasenick for GTP analogs and Y.-K. Ho for helpful

discussions.

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11. Lopez, J. M., A. Dromerick, and E. Freese. 1981. Response of guanosine-5' triphosphate concentration to nutritional changes and its significance for Bacillus subtilis sporulation. J. Bacteriol. 146:605-613. 12. Pace, V., E. Hanski, Y. Salomon, and D. Lancet. 1985. Odorant sensitive adenylate cyclase may mediate olfactory reception. Nature (London) 316:255-258. 13. Racine, F. M., and J. C. Vary. 1980. Isolation and properties of membranes from Bacillus megaterium spores. J. Bacteriol. 143:1208-1214. 14. Sadaie, Y., and T. Kada. 1983. Formation of competent Bacillus subtilis cells. J. Bacteriol. 153:813-821. 15. Shay, L. K., and J. C. Vary. 1978. Biochemical studies on glucose initiated germination in Bacillus megaterium. Biochim. Biophys. Acta 538:284-292. 16. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using

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bicinchoninic acid. Anal. Biochem. 150:76-85. 17. Sonenshein, A. L. 1985. Recent progress in metabolic regulation of sporulation, p. 185-193. In J. A. Hoch and P. Setlow (ed.), Molecular biology of microbial differentiation. American Society for Microbiology, Washington, D.C. 18. Stryer, L., J. B. Hurley, and B. K.-K. Fung. 1981. Transducin: an amplifier protein in vision. Trends Biochem. Sci. 6:245-247. 19. Tanabe, T., T. Nukada, Y. Nishikawa, K. Sugimoto, H. Suzuki, H. Takahashi, M. Noda, T. Haga, A. Ichiyama, K. Kangawa, N. Minamino, H. Matsuo, and S. Numa. 1985. Primary structure of the a-subunit of transducin and its relationship to ras proteins. Nature (London) 315:242-245. 20. Ullah, A. H. J., and G. W. Ordal. 1981. In vivo and in vitro chemotactic methylation in Bacillus subtilis. J. Bacteriol. 145: 958-965. 21. Vary, J. C. 1973. Germination of Bacillus megaterium spores after various extraction procedures. J. Bacteriol. 116:797-802.