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Proc. Nat. Acad. Sci. USA Vol. 70, No. 2, pp. 471-474, February 1973

Adenosine 3':5'-Cyclic Monophosphate as a Regulator of Bacterial Transformation (Hemophilus influenzae Rd/cyclic-AMP assay) EDMUND M. WISE, JR., SUSAN P. ALEXANDER, AND MARILYN POWERS Department of Molecular and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 Communicated by Jack L. Strominger, December 14, 1972 ABSTRACT 3': 5'-cyclic monophosphate from overnight cultures made alkaline and then neutralized as added to exponentially growing cells of Hemophilus in- described by Goodgal and Herriott (24). Purified DNA was fluenzae strain Rd increases competence for transforma- tion 100- to 10,000-fold. Cyclic AMP added to near-station- made essentially after the procedure of Marmur (26). DNA ary or stationary cells does not increase competence over concentrations were measured by chemical (27) or spectro- the high level normally found in the early stationary scopic (28) methods. phase. Transformation Assay. Slant cultures were inoculated into In most bacteria that can take up naked DNA and can in- supplemented BHI broth and were shaken overnight at 37°. corporate of this DNA into its own chromosome, i.e., be In the morning 1% inocula were added to fresh broth and transformed, highest levels of competence for transformation shaken at 37°. Unless otherwise noted, neutralized 1 mM usually occur in late exponential phase and may persist into cAMP was added when the cells were at relative O.D. 0.05 at the stationary phase (1-3). Late exponential or stationary 600 nm on a Coleman Junior II Spectrophotometer (about phase is also the time when highest concentrations of internal true O.D. 0.10); cells were assayed when the relative O.D. or external adenosine 3':5'-cyclic monophosphate (cAMP) in untreated cells had doubled to 0.10. The transformation occur in bacterial strains where this has been measured (4-7). assay mixture contained 0.1 ml of cells (novobiocin resistant), A great variety of effects of added cAMP, especially on catabo- at relative O.D. of 0.1 (about 5 X 107 cells), 0.05 ml of DNA lite repressed functions (8-20), have been seen in wild- solution (2.5 jig DNA from a streptomycin-resistant mutant), type or adenylate cyclase-deficient mutants of several Gram- and 1.0 ml of supplemented BHI broth. The cells were shaken negative strains. No effects of added cAMP have been seen in gently 15 min at 370 and 100 ug/ml of DNase was then Gram-positive species, although cAMP or adenylate cyclase added. After 5 min of additional incubation at 370, 0.1-1.2 have been detected in several Gram-positive species (4, 7, 21- ml of the mixture was added to 10 ml of molten (450) supple- 23). Considering these facts it seemed reasonable to add cAMP mented BHI agar without antibiotic. The cells were then incu- to an exponentially growing culture of a transformable Gram- bated 2 hr at 370 to allow for phenotypic expression and 10 negative strain such as Hemophilus influenzae Rd (24, 25) and ml of molten (450) agar containing 2 mg/ml of streptomycin to look for increased transformability. Our first experiment, was added. 10 Mg/ml of novobiocin was added instead if re- using crude cell lysate DNA, succeeded in showing a 100-fold quired. The plates were further incubated for 20 hr and colo- increase in competence with 1 mM cAMP. Subsequent im- nies were then counted. Results are expressed as transforma- proved techniques yield transformation frequencies up to tion frequency. For this expression viable cells were counted 10,000-fold above the control level. The improvements or recorded optical densities were converted to cell counts. A especially include the use of saturating amounts of partially relative O.D. of 0.1 corresponds to 5 X 108 cells/ml. Trans- purified DNA. formation frequency is transformant count divided by viable MATERIALS AND METHODS cell count. Most of these procedures are minor modifications of those of Goodgal and Herriott (25) or of Alexander and Bacterial Strains. H. influenzae strain Rd of Alexander and Leidy (24). All results were obtained with purified DNA, Leidy (24) was the kind gift of R. M. Herriott. Spontaneous except for the initial experiment described above. mutants of the strain resistant to 1000 Mug/ml of streptomycin Chemicals. cAMP was purchased from Sigma Co., Calbio- or to 5 ,ug/ml of novobiocin (Cathomycin) were selected on chem, or P-L Biochemicals; cyclic GMP (cGMP), hemin, supplemented brain-heart infusion (BHI) agar with the adenine , and NAD were from Sigma Co. Beef antibiotics. Strains were stored on supplemented BHI agar heart 3':5' cyclic phosphodiesterase (29) was a slants or plates at room temperature and were transferred Sigma Co. product. Novobiocin (Upjohn) and streptomycin every 5-7 days. (Schwarz-Mann) were used. Media. Cells were grown on solidified supplemented BHI agar or were added to molten (450) supplemented BHI agar. RESULTS This agar is essentially as described by Goodgal and Herriott (25). Difco brain heart infusion broth was autoclaved and Effects of cAMP on Transformation of Exponentially Grow- sterile hemin (25 Mg/ml) and sterile NAD (2 Mg/ml) were ing Cells. As shown in Fig. 1, additions of cAMP as low as 0.1 added to the cooled broth. 1% agar was added as required. mM resulted in a marked increase in transformation frequen- The doubling time of H. influenzae Rd in liquid supplemented cies of exponentially growing H. influenzac Rd. Transforma- BHI medum was 35-40 min at 37°. tion frequencies in this strain in exponential phase are at least 10-fold higher than spontaneous rates for strepto- DNA Preparation. Crude DNA preparations were made mycin resistance as we measure these functions. In the pres- 471 Downloaded by guest on October 2, 2021 472 : Wise et al. Proc. Nat. Acad. Sci. USA 70 (1973)

Fig. 2A also shows a small decrease in competence in un- treated cells before the subsequent large increase as the cells approach stationary phase. This decrease was not always seen. It does not appear to be caused by highly competent cells left over from the overnight inoculum (data not shown). There is also a decrease in competence of the cAMP-treated cells be- fore stationary phase. This effect is always seen. Increasing the 0 z cAMP concentration to 2 mM does not alter this decrease. As w a shown in Fig. 2B, if cAMP is added at a later time in the growth w LL cycle the drop is not seen. With both cAMP-treated and con- z trol cells competence soon decreases in stationary phase, al- 0 10.0 I though in the experiments shown in Fig. 2A and B control 4 CD)z cells were not monitored long enough to show this. 0 J LU. m added at up to 5% concentration has only minor U') . 0 z 1.0 a effects on growth rate or on competence (data not shown). r LIi Presumably in this rich brain-heart infusion medium repres- 4 cr sion of competence resulting from low concentration of cAMP, and possibly other means, is so high that even high glucose 0.I concentrations have little further effect on cAMP concentra- LI tions and, therefore, on competence. Other experiments de- signed to show a strong glucose effect in the presence or ab- sence of cAMP showed little or no effect of glucose in this medium. cAMP CONCENTRATION (mM) Time Course of cA MP-Stimulated Increase of Competence. FIG. 1. Transformation frequency and growth rate with in- As shown in Table 1 competence is not immediately increased creasing cAMP concentration. cAMP was added at relative O.D. by cAMP addition, but requires at least one-half generation 0.05. Transformation frequency (@-@) was monitored 30 min after cAMP addition (see Methods). Doubling time 1 hr after cAMP addition is also shown (0----O).

ence of 1 mM cAMP the normal transformation rate is in- creased over 10,000-fold. Peak competence is obtained with 5 mM cAMP. These findings suggest that the transformation frequency increase could be used as a quantitative assay for a lo-5. cAMP in quantities as low as 5 nmol. La. Fig. 1 also shows the growth rates at the end of the first hour after addition of cAMP. 1 mM cAMP always shows an 41 immediate 10-20% slowing of growth rate; this slowing per- c ro-6 0 Ui. sists to the stationary phase. 10 mM cAMP shows a gradually C') z increasing effect on growth rate such that in 3 hr growth ceases 4 even though the total growth may be only 30% of maximum Hr stationary phase O.D. without added cyclic AMP (data not shown). In these experiments cAMP was added to exponen- tially growing cells. If 10 mM cAMP is added to a culture flask freshly inoculated with overnight culture a slight amount of growth takes place initially; then, there is stasis with some lysis (data not shown). No effect of 1 mM cAMP on lysis in- duced by cold or deoxycholate was seen in highly transform- z able cells prepared as described at the beginning of this para- -J graph. 4 0 P a. Competence During the Growth of the Culture. In H. influenzae 0 Rd competence is greatest in late exponential and early sta- tionary phase as shown in Fig. 2A, and as shown by others (3). This figure shows that 1 mM cAMP added in exponential Minutes phase and present during all subsequent growth of the culture FIG. 2. cAMP effect on competence at different stages of increases competence as much as 4000-fold, but at no point is growth. (A): 1 mM cAMP was added at arrow. Transformation without cAMP or with cAMP-induced competence than normal frequency (0 0) cAMP (0-O), higher stationary and relative O.D. of the culture without cAMP (0 0) or lhase competence attained in the absence of added cAMP. with cAMP (0 O) are plotted as function of time. (B): Presumably, the endogenous internal or external cAMP con- similar to (A) except that cAMP was added when the culture centration is adequate in stationary phase to have maximum was at a higher O.D. In (A) and (B), cells were in exponential competence without added cAMP. growth before addition of cAMP. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) cAMP as a Regulator of Bacterial Transformation 473

for an appreciable increase. This long time suggests that a TABLE 1. Time course of competence increase major change occurs in the cell. Transformation in Agar Stimulated by cAMP. Fig. 3 shows Time after that if cAMP is present in agar and additional agar containing cAMP Time/ Transformation Frequency low-competence exponential cells l)lus DNA is poured on top added Generation of the cAMP layer, increased competence is seen. There are (mil) Time -cAMP +1 mM cAMP 200 times more transformants per unit area in the center (where 0 0 0 0 the cAMP was spotted) than in the periphery. This experiment 8 0.2 0 can be reversed (not shown) and cAMP can be spotted on 15 0.4 0 2X10-8 solidified agar containing low-competence cells plus DNA. 30 0.8 0 5 X 10-6 This procedure can be adapted to a simple semi-quantitative, 45 1.2 1 X 10-8 4 X 10-5 or possibly quantitative, assay for cAMP. 60 1.6 3X10-8 8X10-5 Additional Experiments. cAMP obtained from three differ- DNase was added to the transformation mixture after 3 min ent suppliers gave similar results oln competence and growth instead of the usual 15 min. Zero means <1 X 10-8; 8-min cells rate. Ashed cAMP had no effect oln either function. Several without cAMIP were not assayed. The generation time is that of other substances were tested for their ability to increase com- the control culture. The cAMP-treated culture grew about 15% petence and none of these were effective. These included N, 0- slower. The O.D. at zero time was 0.05. By 60 min, control cells dibutyryl cAMP, 1 mM; 3':5'-cyclic monophos- have begun their normal rise in competence. l)hate (cGMP), 1 and 5 mM; , 1 and 5 mM; and 5'-AMP, ADP, and ATP, 1 mM. cAMP treated with 3': 5'- phoslphodiester- To see if the mere slowing of growth rate by cAMP were the ase before addition to exponential cells lost its ability to in- cause of the increased competence, a series of toxic agents were crease competejice. This is an additional proof that it is not added in concentrations that gave immediate 10-30% inhibi- some minor comlponent in commercial cAMP that increases tion of growth. These included 0.1 mM cadmium acetate, competence. 1 mM cupric chloride, 0.01 mM mercuric chloride, 1 mM manl- Most of our work involved the introduction of DNA con- ganous chloride, and 1 mM sulfanilamide. No increase in com- taining a streptomycin resistance marker into streptomycin- petence was seen with any of these agents. sensitive, novobiocin-resistant cells. Similar effects of cAMP

FIG. 3. cAMP induction of competence in agar. 1 pmol of dry sterile cAMP was spotted near the center of the plate containing 5 ml of supplemented BHI agar and was allowed to diffuse 18 hr; then 2.5 ml of agar containing 107 exponential cells and 10ljg of DNA was poured over the plate and the plate was incubated at 370; 3 hr later, 200 sg of DNase was added in 2 ml of agar; 1 hr later, 5 ml of 0.1% streptomycin agar was added and the plate was incubated 2 days at 37°. Downloaded by guest on October 2, 2021 474 Biochemistry: Wise et al. Proc. Nat. Acad. Sci. USA 70 (1973)

on competence were seen when we introduced novobiocin This work was supported by grant CA-08982 from the National from The American Heart DNA into streptomycin-resistant, novo- Cancer Institute and grant 71,908 resistance-marked Association. biocin-sensitive bacteria (data not shown). It is true that is linked to novobiocin resistance in streptomycin resistance 1. Anagnostopoulos, C. & Spizizen, J. (1961) J. Bacteriol. H. influenzae Rd (30, 31), but this should not have any im- 81, 741-746. portant bearing on our results. 2. Tomasz, A. (1969) Annu. Rev. Genet. 3, 217-232. 3. Hotchkiss, It. D., & Gabor, M. (1970) Annu. Rev. Genet. DISCUSSION 4, 193-224. From this work it appears that transformation in H. influenzae 4. Okabayashi, T., Yoshimoto, A. & Ide, MI. (1963) J. Bacteriol. Rd can be added to the list of functions regulated by cyclic- 86, 930-936. 5. Makman, R. S. & Sutherland, E. W. (1965) J. Biol. Chem. cAMP-mediated functions in bacteria or AMP. The known 240, 1309-1314. bacteriophages are denoted catabolite-repressible functions; 6. Peterkofsky, A. & Gazdar, C. (1971) Proc. Nat. Acad. Sci. most involve fairly directly control of the utilization of energy, USA 68, 2794-2798. carbon, or nitrogen sources on the part of these bacteria or 7. Clark, V. L. & Bernlohr, l'. W. (1972) in Spores V, ed. cAMP of transformation is hard to fit into Halvorson, H. O., Hanson, It. & Campbell, L. L. (American phages. regulation Society for Microbiology, Washington, D.C.), p. 167-173. proven transformation in such a pattern. We have not that 8. Magasanik, B. (1970) in The Lac , ed. Beckwith, Hernophilus is a function that catabolites directly repress in J. it. & Zipser, 1). (Cold Spring Harbor, New York), p. either a transient or a permanent manner. The effects of added 189-219. cAMP that we show are not immediate ones, and this delay 9. Monard, D)., Janecek, J. & ltickenberg, H. V. (1969) Rio- chem. Biophys. Res Commun. 35, 584-591. could mean that there is a massive change or changes in the 10. Pastan, I. & Perlman, It. (1970) Science 169, 339-344. cell, possibly similar to those causing the buoyant density de- 11. Pastan, I. & Perlman, it. L. (1971) Nature New Biol. 229, crease seen in competent Bacillus subtilis cells. We have not 5-8. proven that cAMP is a, or the, normal signal for competence 12. Perlman, lv. L. & Pastan, I. (1971) in Current Topics in E. R. turn-on, but alternatives to this are unlikely. True proof would Cellular Regulation, ed. Horecker, B. L. & Stadtman, (Academic Press, New York), Vol. 3, p. 117-134. may be difficult to ob- require adenylate cyclase mutants that 13. Dobrogosz, W. J. & Hamilton, P. B. (1971) Biochem. tain in Hemophilus species, since they are fastidious and do not Biophys. Res. Commun. 42, 202-207. utilize many sugars. 14. Harwood, J. & Smith, 1). H. (1971) Biochem. Biophys. Res. Catabolite repression is at least partly mediated by cAMP, Coinrnun. 42, 57-62. W. P. & Beeman, I). K. (1971) Biochem. that should not be considered the whole story. Just as the 15. Hempfling, but Biophys. Res. Commun. 45, 924-930. lactose operon in requires an inducer as well as 16. Hong, J. S., Smith, G. It. & Ames, B. N. (1971) Proc. Nat. the permissive action of cAMP, transformation may have Acad. Sci. USA 68, 2258-2262. similar or even greater complexities. We can get competence 17. Kuwano, M. & Schlessinger, 1). (1970) Proc. Nat. Acad. levels of exponential cells up to stationary cell competence Sci. USA 66, 146-152. T., Arditti, It. It. & Eisen, HI. (1972) Proc. Nat. followed by some 18. Grodzicker, level, but with an anaerobic growth period A cad. Sci. USA 69, 366-370. aerobic growth others have shown that competence is even 19. Nakazawa, A. & Tamada, T. (1972') Biochein. Biophys. greater than found in our experiments (25). This may imply Res. Commun. 46, 1004-1010. that other inducers of transformation are necessary. For in- 20. Nealson, K. H., Eberhard, A. & Hastings, J. W. (1972) USA 69, 1073-1076. stance an unknown factor of this sort has been recovered from Proc. Nat. Acad. Sci. 21. Hirata, M. & Hayaishi, 0. (1967) Biochian. Biophys. Acta Hemophilus cultures (32). It will be of interest to see if this 149, 1-11. factor is cAMP. 22. Khandelwal, it. L. & Hamilton, I. It. (1971) J. Biol. Chem. cAMP may have a role in transformation in other bacteria, 246, 3297-3304. 262-268. but this may be harder to prove because of factors such as 23. Ide, M. (1971) Arch. Biochent. Biophys. 144, 24. Alexander, H. E. & Leidy, G. (1951) J. Ex-p. Med. 93, 345- cAMP permeability. Our laboratory and others have attempted 359. in vain to correct diauxie curves or induce sporulation in 25. Goodgal, S. H. & Herriott, It. M. (1961) J. Gen. Physiol. (transformable) Bacillus species by adding cAMP to expo- 44, 1201-1227. Diauxie, probably universally, is a 26. Marmur, J. (1961) J. Mfol. IBiol. 3, 208 -218. nentially growing cultures. ed. catabolite-repression phenomenon involving cAMP, although 27. Schneider, W. C. (1957) in Methods in Enzymology, Colowick, S. P. & Kaplan, N. 0. (Academic Press, New there may also be competition for substrate entry. Sporulation York), Vol. 3, p. 680-684. is probably also a catabolite-repressed phenomenon (7, 33, 34), 28. Hotchkiss, 1'. D). (1957) in Methods in Enzynmology, ed. involving at least in part cAMP. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New Competence stimulation by cAMP in Hemophilus is pre- York), Vol. 3, p. 708-715. R. W. & Sutherland, E. W. (1962) J. Biol. Chem. as are cAMP 29. Butcher, sumably at the transcriptional level, almost all 237, 1244-1250. effects in bacteria (8, 35, 36). But whether the effect is di- 30. Goodgal, S. H. & Herriott, It. M\4. (1957) 42, 372- rectly on the of one or more components of the 380. transformation system or is more indirect, such as on the 31. Stuy, J. H., Ph.D). Thesis, University of Utrecht, Nether- transcription of message for a modifier of RNA poly- lands, 1961. 32. Barnhart, B. J. (1967) Biochim. IBiophys. Acta 142, 465- merase or for a sigma factor, is unknown. Additionally, the 474. high cAMP concentrations that exist in transformable and 33. Elmerich, C. & Aubert, J. P. (1972) Biochcein. Biophys. Res. nontransformable strains as the cells either approach sta- Commun. 46, 892-897. & P. Proc. Nat. tionary phase or are in stationary phase may have more signifi- 34. Schaeffer, P., Millet, J. Aubert, J. (1965) The growth inhibition Acad. Sci. USA 54, 704-711. cance than just to aid transformation. 35. Varmus, H., Perlman, R. & Pastan, I. (1970) J. Biol. Chem. by exogenous cANP in Hemophilus may be telling us some- 245, 2259-2267. thing very important about stationary phase and cAMP that 36. Eron, L., Arditti, it., Zubay, G., Connaway, S. & Beckwith, is applicable to bacteria in general. J. W. (1971) Proc. Nat. Acad. Sci. USA 68, 215-218. Downloaded by guest on October 2, 2021