Adenosine 3':5'-Cyclic Monophosphate As a Regulator of Bacterial Transformation (Hemophilus Influenzae Rd/Cyclic-AMP Assay) EDMUND M
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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 Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 Communicated by Jack L. Strominger, December 14, 1972 ABSTRACT Adenosine 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 genes 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 nucleotides, and NAD were from Sigma Co. Beef antibiotics. Strains were stored on supplemented BHI agar heart 3':5' cyclic nucleotide 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 mutation 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 Biochemistry: 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 Glucose 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.