JOURNAL OF BACTERIOLOGY, May 1979, p. 314-319 Vol. 138, No. 2 0021-9193/79/05-0314/06$02.00/0 Specific Inhibition of Outgrowth of Bacillus subtilis Spores by Novobiocin MARK GOTTFRIED,t CRISTIAN ORREGO,* ALEX KEYNAN,4 AND HARLYN 0. HALVORSON Department ofBiology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154 Received for publication 17 February 1979 Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive tem- perature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while dis- playing a marginal effect on logarithmic growth during one generation time. Inhibition ofoutgrowth by novobiocin occurred in the absence ofDNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se. Ginsberg and Keynan (15) confirmed earlier Growth of bacteria. LB agar plates contained (in observations (6, 24, 26, 28, 33) pointing to the grams per liter): tryptone (Difco), 10; yeast extract independence of Bacillus subtilis spore out- (Difco), 5; NaCl, 5; and agar, 15. Soft agar for phage growth from DNA synthesis. They demon- overlay contained (in grams per liter): nutrient broth 168 (Difco), 8; NaCl, 5; and agar, 7. Vegetative growth, strated that outgrowth of B. subtilis pro- sporulation, outgrowth, and preparation ofspores were ceeds normally under restrictive conditions in as described elsewhere (26) with the exception that mutants temperature sensitive in DNA synthe- the final step of purification on a discontinuous Ren- sis. Two inhibitors of DNA synthesis, nalidixic ografin gradient was eliminated. The purified spore acid and 6-(p-hydroxyphenylazo)uracil, im- stocks contained between 82 and 97% of the spores in paired outgrowth. However, the inhibition of the phase-bright state. Spore stocks were checked for outgrowth and not that of DNA synthesis is the expected genetic markers and heat resistance by reversed by glucose (15). plating spores after heating for 10 min at 800C. Spores In probing the generality of these observa- were not heat activated before transfer into the broth. Changes in optical density were followed in a Klett- tions, we were surprised to observe that the Summerson photoelectric colorimetric equipped with antibiotic novobiocin, also a DNA synthesis in- red filter no. 66. hibitor, completely impaired outgrowth even un- Nucleic acid synthesis. Synthesis of DNA and der glucose-enriched conditions. This drug inter- RNA were measured by incorporation of [3H]thymi- acts with and inhibits the enzyme DNA gyrase dine and ['4C]uracil, respectively (final concentration (Eco DNA topoisomerase II) responsible for in- in the culture, 5 ACi/ml), into trichloroacetic acid- serting negative superhelical twists into the insoluble material (26) in culture samples containing Escherichia coli chromosome (9, 13). This paper at least 108 exponentially growing cells. For outgrowth, demonstrates that novobiocin at a concentration 5 ,uCi of [3H]thymidine or 0.625 ,Ci of ["4C]uracil per inter- ml was used. DNA was also estimated by the colori- that completely inhibited DNA synthesis metric method of Burton (2). fered with outgrowth but interfered substan- Genetic transformation. Competent cell cultures tially less with exponential growth. of B. subtilis 168 trpC2 were prepared by the method of Bott and Wilson (1). Purified donor DNA (10 ytg) MATERLALS AND METHODS (23) from strain C0169 was mixed with 1 ml of com- Bacteria and phages. The genotypes of bacterial petent cells for 30 min at 37°C. Cells were then col- and phage strains used are given in Table 1. lected by centrifugation, washed with 5 ml of Penassay in 10 ml of the same t Present address: Rosenstiel School of Marine and Atmos- broth (PAB), and resuspended pheric Science, University of Miami, Miami, FL 33149. medium. Two generations of growth were then per- t Present address: Section of Development and Molecular mitted to obtain marker segregation. Cells were con- Biology, Institute of Life Sciences, The Hebrew University of centrated by centrifugation and spread on LB plates Jerusalem, Jerusalem, Israel. containing 5 jig of novobiocin per ml. 314 VOL. 138. 1979 SPORE OUTGROWTH AND NOVOBIOCIN 315 TABLE 1. Strains used Strain Genotype Source B. subtilis 168 trpC2 Our stock B. subtilis ANS-1 Spontaneous trp revertant of 168 Our stock B. subtilis BD54 keuA8 metB5 ile-1 D. Dubnau B. subtilis BD43 dnaC metB5 ike-1 dnaC30 D. Karamata (19) B. subtilis C0169 trpC2 thyA thyB novAl N. Sueoka (16) B. subtilis 168 novA trpC2 novAl Our stock B. subtilis BC50 ade-16 keuA8 metB5 nic J. Copeland B. licheniformis ATCC 8480 American Type Culture Collec- tion Phage AR-9 A. A. Prozorov via D. Dubnau Transduction. Transduction was used to ascertain system was completely suppressed by novobio- the genetic location of the nov marker transferred into cin (Fig. 10). Its addition to a vegetative cell strain 168. AR-9 phage was titrated by infection of 0.1 culture brought an immediate inhibition ofDNA ml of a Bacillus licheniformis culture in PAB (at a Similar re- cell density of 60 to 80 Klett units). After 10 min at synthesis (Fig. 1D). experiments 370C, the suspension was spread onto M agar plates vealed that 1.7 ,ug of novobiocin per ml had no (34) with a 2.5-ml soft agar overlay. Distinct plaques effect on spore outgrowth (data not shown). appeared after about 20 h of incubation at 37°C. Outgrowth is not dependent on DNA rep- Transducing lysates were obtained by infecting 0.1 lication. Ginsberg and Keynan (15) demon- ml of the donor strain culture containing the nov strated that the outgrowth process occurred al- marker (5 x 108 cells per ml) with phage at a multi- most normally in mutants conditional for DNA plicity of infection of 1 and allowing 10 min of incu- replication under nonpermissive conditions. bation before transfer of the mixture to an M plate by Spores of a thymidine-dependent mutant were the soft overlay method. After 20 h at 27°C, the plate in the absence of was flooded with 5 ml of phage buffer (8), and the top also capable of outgrowth layer of the plate was removed with a sterile spatula. thymidine (15). Both of these experiments were The plate was further rinsed with 5 ml ofphage buffer. performed in defined media. The same results The collected suspension was centrifuged, and the were obtained in rich medium. Germination ki- agar particles were discarded. The resultant superna- netics and the time of cell emergence were the tant fraction was filtered through a membrane filter same for a mutant temperature sensitive for (Millipore Corp.; type HA, 0.45-,um pore diameter). DNA synthesis and its isogenic parent (Fig. 2A Titers of phage stocks varied from 5 x 109 to 5 x 101 and B). Novobiocin prevented outgrowth in both plaque-forming units per ml. cultures. Thus, novobiocin sensitivity was not Transduction was performed by inoculating a cul- ture of the recipient strain BC50 in PAB at 20 Klett dependent upon DNA replication. units, followed by 5 h ofincubation at 37°C with rotary Time of escape from inhibition of out- agitation. At this time cultures displayed cells of high growth by novobiocin. Addition of the anti- motility and were then infected with the transducing biotic 25 min after the initiation of germination phage at a multiplicity of infection of 1. This was still prevented subsequent increase in cell mass followed by gentle agitation for 15 min. Cells were (Fig. 3). Germination was well completed by 20 collected by centrifugation, washed with mineral salts min. Loss of sensitivity of the system to the medium (26), and plated on selective medium. addition of novobiocin began to occur at 60 min, Chemicals. Novobiocin and nalidixic acid were approximately coincident with the time of initi- purchased from Sigma Chemical Co. Rifampin was a product of Schwarz/Mann. [CH3-3H]thymidine (6.7 ation of DNA replication (Fig. 10), implying Ci/mmol) and [2-'4C]uracil (57 mCi/mmol) were ob- that replication released the germinated spore tained from New England Nuclear Corp. from novobiocin inhibition. RNA synthesis in the presence of novo- RESULTS biocin. Addition of the antibiotic at 25 min of Effect of novobiocin on spore outgrowth. outgrowth resulted in a 63% decrease in accu- The presence of 4.6 ,ug of novobiocin per ml had mulation oflabeled RNA within the next 35 min no effect on the germination ofB. subtilis spores (Fig. 4A). Exposure of exponentially growing but completely inhibited their outgrowth (Fig. cells to novobiocin reduced RNA synthesis by 1A). The same concentration of the antibiotic, essentially the same degree (Fig. 4B). Rifampin when introduced at a midpoint during exponen- (8 ,ug/ml) immediately inhibited RNA synthesis tial cell multiplication, displayed a marginal ef- in both cultures when introduced at the same fect on the increase of turbidity during one gen- time as novobiocin (data not shown). eration time (about 20 min) (Fig. 1B). The first Novobiocin resistance mutation removed cycle of DNA replication in the outgrowing cell the novobiocin effect during outgrowth. 316 GOTTFRIED ET AL. J. BACTrERIOL. Sporulation occurred normally in the A B presence of novobiocin. A number of inde- pendent studies have determined that chromo- some replication is completed within 2 h after _ / l logarithmic growth ceases (10, 21) and that chro- - l0o/ mosome termination is a prerequisite for the completion of sporulation (10, 21, 22).