Factors Affecting Bacterial Competence

Transcription

1 BACTERIOLOGICAL REVIEWS, Dec. 1968, p Copyright 1968 American Society for Microbiology Vol. 32, No. 4, Pt. 1 Prinited in U.S.A. Factors Affecting Bacterial Competence for Transfection and Transfection Enhancement H. T. EPSTEIN Departmenzt of Biology, Brandeis University, Waltham, Massachusetts 2154 INTRODUCTION MATERIALS AND METHODS RESULTS DiSCUSSION LITERATURE CITED INTRODUCTION There is at present no real insight into the physiological nature of the competent state of bacteria. Indeed, it is not even known whether there is a single competent state or whether competency depends on factors other than bacterial properties. Accordingly, it appeared desirable to study competency for two different deoxyribonucleic acid (DNA) preparations in the same kind of competent cells. This can be done by studying transformation and transfection in one bacterial species or by studying two kinds of transformation or of transfection in one bacterial species. Bacillus subtilis exhibits competencies for transformation and for two kinds of transfection. The present work is a comparative study of the two kinds of transfection. Transfection in B. subtilis is characterized (7, 15-17) by two different dependencies on phage DNA concentration. For example, SP2 transfects linearly with DNA concentration (15), and SP82 transfects as the third or fourth power of the DNA concentration (7). For SP82, Green (7-9) has supplied evidence that the nonlinear class results from inactivation of incoming phage DNA, which makes genetic recombination necessary for the production of replicating phage molecules. SP82 DNA, moreover, differs from host DNA in that it contains hydroxymethyluracil in place of thymine [Kahan and Kahan, quoted by Green (7)]. Thus, it is "foreign" DNA and would be expected to be attacked by defensive cellular nucleases. Experiment and expectation agree in the inactivation of the SP82 DNA. It is, then, surprising that the linearly transfecting SPO2 DNA actually (15) transfects less efficiently per microgram of DNA than does SP82 DNA. Further, SP2 DNA has a molecular weight approximately 25% of that of SP82 DNA, judged from X-ray inactivation curves obtained in our laboratory. One would expect this smaller DNA to be extracted and purified in appreciably less damaged form than the DNA of SP82. SPO2 DNA is also likely to be homologous to host DNA, because SPO2 appears to be a temperate phage (15) and because SPO2 DNA and host DNA have the same buoyant density in CsCl (15). In contrast to the expectations raised by this comparison of SPO2 DNA and SP82 DNA, one finds that, in the same batch of competent cells, a less damaged and apparently homologous DNA transfecting linearly with DNA concentration is markedly less efficient in transfection than a more damaged, heterologous DNA requiring genetic recombination for plaque formation. The situation is presented visually in Fig. 1, which also shows the enhancement and linearization of SP82 transfection as a result of pre-exposure of competent cells to ultraviolet (UV)-irradiated Escherichia coli DNA (5). After enhancement, the linear SP82 transfection is 1-fold greater than SP2 transfection. There are two quite different kinds of explanations for the low SP2 DNA transfection: (i) SP2 may replicate poorly in competent host cells or (ii) lysogenization may have occurred with the consequent loss of infective centers. The first explanation starts from the abovementioned observation that SPO2 DNA is homologous to host DNA by the buoyant density criterion. It is also known (3, 13) that DNA synthesis is arrested in competent cells. Presumably, there has been a sequestering of some enzyme(s) needed for DNA synthesis. Because of the homology of SPO2 DNA to host DNA, there may be a decreased opportunity for replication of SPO2 DNA, which needs to use host enzymes for such purposes. During the period of arrested DNA synthesis, the SP2 DNA could be slowly degraded by host nucleases, thereby decreasing 313

2 314 EPSTEIN BACTERIOL. REV. o vi nts E 14-, 2I -~ z zul (n z W- 13: A I tj- 12-5XI OI,/A'4 / /1 / / /, / // I // / 2 1 I / 3/ /I /3/ SP82 ENHANCED I* I, I r, I, I.. O. 3 DNA CONCENTRATION -/zg/ml. // FIG. 1. Dependence of plaque formation on concentration of SP2 and SP82 DNA. Also shown is the SP82 transfection enhancement by 1.5,ug of Escherichiia coli DNA given about 14 ergs/mm2 of ultraviolet irradiation from a sterilamp. The dashed lines show the theoretical first, second, and third power dependencies on DNA conicenitration. the effective transfecting capacity. The sequestering could be largely irrelevant to SP82 DNA because this large phage has the same molecular weight of DNA as coliphage T4 (7), which carries or induces enzymes essential for its own replication, especially its own DNA polymerase and ligase (1, 6; B. Weiss, T. R. Live, and C. C. Richardson, Federation Proc., 62:395, 1967). Another result relevant to the experiments to be described is the finding (7, 17) that recombination frequencies are much higher for transfection crosses than for the same crosses done with intact phages. This could be the consequence of at least two entirely different mechanisms. First, as set forth above, competent cells may contain increased levels of nucleases attacking foreign DNA such as the hydroxymethyluracilcontaining DNA from phage SP82. Second, transfections are usually done with a DNA concentration (1 to 2,ug/ml) equivalent to 25 to 5 molecules of intact DNA per cell. If this value reflects DNA uptake in the competent cells in the culture, transfection corresponds to what may be termed an "instant pool" of phage DNA molecules. It is known that phage recombination frequencies increase with multiplicity of infection (14) and with time in the latent period (2, 12). The postulated increase in nuclease attack on SP82 DNA should result in transfections which decrease in level with the length of time the precompetent cells have grown in competency medium. This conclusion is at variance with the fact that, during that growth period, transfection first increases to a maximum before decreasing. Therefore, there must be a factor which increases competence at early times. The literature contains two findings which may explain the increase in competency. First, there may be an increase in ability to fix DNA irreversibly, since competency generally follows the ability to fix DNA (11). Second, Wolstenholme et al. (19) recently showed that the number of mesosomal sites per cell increases as competency develops in B. subtilis. In addition, they showed an association of radioactively labeled DNA with these sites. If mesosomal sites are thus identified with DNA-replicating sites, incoming DNA should find a progressively increasing chance of becoming associated with a replicating site as the number of mesosomal sites increases. Then, once the number of sites has reached its maximum, competence should remain constant unless it decreases due to nuclease attack on the incoming DNA. MATERIALS AND METHODS All media used and the methods of preparation of phages, competent bacteria, and the various types of DNA have been described in detail previously (4, 5). The bacterium used in all experiments was B. subtilis strain SB1 (requiring histidine and tryptophan). Competent cells were obtained by diluting frozen precompetent cells 1-fold into competency medium and incubating them with shaking for various times. These cells are described as, e.g., 6-min competent cells if the incubation lasted 6 min. Transfections were generally done by adding.1 ml of DNA solution to.5 ml of competent cells. Assays for plaque formation were done after 5 min of incubation at 37 C. DNA concentration in purified samples was determined by multiplying the optical density at 26 nm by 5 to obtain the result in micrograms per milliliter. DNA in nonpurified samples was determined by the Ceriotti method, with purified SP82 DNA as the standard. RESULTS The initial studies were of the two explanations suggested for the low level of SPO2 transfection:

3 VOL. 32, 1968 FACTORS AFFECTING BACTERIAL COMPETENCE 315 TABLE 1. Plaque formation by phages adsorbed to 2 X 18 to 4 X 18 cells/ml 15 F Prepn SP2/ml Input phages X 15 Phages preadsorbed to Noncompetent cells X 15 6-min competent cells X 14 Phages incubated 2 min in Competency medium X 15 "Conditioned" competency medium X 15 Phages preadsorbed to 6-min competent cells, centrifuged, and resuspended in broth Pellet X 14 Supernatant fluid....12x 14 SP82/ml K 2.5 X 15 1 E X 15 a, 1L X X 15 (i) poor replication of SPO2 in SB1 due to sequestering of enzymes associated with synthesis of host DNA and (ii) loss of infective centers because of lysogenization by SPO2. The first experiment simply measured the efficiency of plating after adsorption of phages on broth-grown SB1 cells and on precompetent SB1 cells grown in competency medium for various times. To 1-ml amounts of such cells were added.1-ml amounts of a phage suspension containing about 18 phages/ml. After 2 min for adsorption, the suspensions were diluted and assayed for plaque-forming units. Table 1 contains data from typical experiments. SP82 phages had the same titer whether preadsorbed to 6-min competent cells or plated directly on seed cells. However, SPO2 phages had a 9-fold lower titer when preadsorbed to 6-min competent cells than when plated directly on seed cells. Similar experiments showed that SP2 plaque formation dropped from about 5% when preadsorbed to -min competent cells to 17% on 3-min cells, 11 %7o on 5-min cells, and 5% on 8-min cells. Controls shown in Table 1 were done to check TABLE [ Input Phages ~~~~~~ - <~ ~~~~* * Incubation Time (min) 2.1 X 15 FIG. 2. Plaque formers present in a suspension of.4 X 15 6-min competent cells at various times after addition of about 13 SP2 phages. adsorption and the effect of incubating phages in fresh competency medium and in "conditioned" competency medium (that remaining after SB1 cells had been made competent therein for 9 min, the cells then having been removed by centrifugation). The kinetics of the loss of plaque-forming ability were measured by plating portions of an SPO2 phage-infected culture of 6-min competent cells. The results of such an experiment (Fig. 2) show that a very rapid loss of plaque-forming ability occurred in the first 2 min after the cells and phages were mixed. If the loss of infective centers is due to lysogenization, the bacteria should survive the infection. At low multiplicities of infection, the surviving cells cannot be measured directly, but at multiplicities somewhat greater than unity there should be a measurable increase in number of survivors above those killed by the adsorption of phages. Table 2 shows the results of an experiment in which 75-min competent cells were incubated with various multiplicities of SPO2 phages for 2 Bacterial and SP2 phage survival in infected 75-min competent cells (A) (C) (D) (E) (F)(G Experimental (B) Expected Experimental Input Plaques(G) infection e- (A) bacteria bacteria put foues (F)/(E) multiplicity surviving surviving phages found X X X X X X X 18.2 X X X X 18.8 X X X 1.64 X 1.2 X 18.31

4 316 EPSTEIN BACTERIOL. REV. min before being plated for plaque formation and for colony formation. Adsorption of phages by such competent cells was always rapid and was essentially 1%/- within 5 min. At a multiplicity of 3.7, the expected number of surviving bacteria was equal to the experimental number, which was.25 of the starting number of bacteria. The efficiency of plating of the input phages was.67, in agreement with figures presented earlier. If the missing phages had lysogenized the bacteria, the number of surviving cells would have greatly exceeded the number of cells actually found. A similar conclusion may be drawn from the data obtained with a multiplicity of At lower multiplicities, the effect could not be detected even if it existed, but the loss of plaque formation by the input phages was roughly constant for all multiplicities. The postulated sequestering of DNA synthesis enzymes was next tested by obtaining one-step growth curves for the two phage strains, both by infection with viable phages and by transfection with phage DNA (Fig. 3). A typical curve was obtained for SP82 phages adsorbed on 9-min,1 a) D 16 I 123 lo2: \1~1 /"-- / o \ 3P82 DNA SP2 DNA SP2 PHAGES SP2 PHAGES INCUBATION TIME (MINUTES) FIG. 3. Plaque -formers present in a suspension of 9-min competent cells at various times after addition of SP2 or SP82 phlages or their DNA. At 2 min after 1,ug ofeach type ofdna was added, further DNA uptake was stopped by additiont of 2,ug of deoxyribonuclease. *Is competent cells. The latent period of about 4 min was the same as that found by Green (7), and in our laboratory, for infection of broth-grown cells. The burst size was more than 1. SP82 DNA transfection yielded a one-step growth curve similar to that for phage infection except for the increased latent period typically found for transfection (7, 18). However, after exposure of 9-min competent cells to SPO2 phages or SPO2 DNA, there was no increase in plaque formation for at least 3 hr after infection was initiated. In our laboratory, the latent period for SPO2 phage infection of broth-grown cells was about 32 min, with a burst size of about 1. A single experiment on SPO2 phage infection of -min competent cells also gave no burst for more than 3 hr. The slow decrease of SPO2 plaque formation indicates a slow inactivation of the phage DNA. The lysogenization interpretation was further tested by obtaining growth curves for SPO2 phages and SPO2 DNA on competent cells which had been irradiated for 3 min, a UV dose which maximizes transfection enhancement for phage SP82 (4). If lysogenization were the cause of loss of infective centers, the irradiation might be expected to inhibit lysogenization and drive the phages into the lytic cycle. However, growth curves on such irradiated cells showed no transfection enhancement and no increase in plaque formation for at least 3 hr. It seems probable that there is a sequestering of enzymes needed for SPO2 growth in competent cells, and that formation of the needed enzymes is induced by SP82 phages or phage DNA infecting samples of the same competent cells. Evidence was next sought for the hypothesized nucleases attacking the incoming transfecting DNA. If the enzyme level changes, the extent of inactivation of the DNA should change correspondingly. This deduction was tested by measuring the number of transfectants as a function of the phage DNA concentration. Transfecting DNA was added to cells after 35 min and after 8 to 9 min of incubation in competency medium. Plaque formation was assayed after an additional 5 min of incubation. As shown in Fig. 4, SP82 DNA transfection had a greater dependence on DNA concentration on 9-min competent cells than on 35-min competent cells. Therefore, there was a greater level of nucleases at the later time. SPO2 DNA transfection showed the opposite pattern: greater transfection dependence on 35- min competent cells. Plaque formation was nearly linear on the 9-min competent cells, indicating that there was little attack on the DNA at that time. Presumably, then, at 35 min an enzyme is present which can attack SPO2 DNA, and this

5 1 3 :, ~ z- _6~ VOL. 32, 1968 FACTORS AFFECTING BACTERIAL COMPETENCE 317 ; 14 Ion C:) 12 z -o - o }s9 DNA/ML FIG. 4. Dependence of transfection on DNA concentration for phages SP2 and SP82 incubated witht 35- anid 9-min competent cells. Dashled lines as in legend for Fig. 1. enzyme is subsequently sequestered along with the DNA-synthesizing enzymes, as would be expected for cells preparing to take in DNA homologous to that of the host. The 1-fold higher level of SP2 transfection on the 35-min competent cells indicates an earlier development of maximal competency for this phage DNA. This point was studied directly by measuring the transfection with 1,ug of phage DNA on cells incubated for increasing times in the competency medium. The cells were exposed for 5 min before plaque formation was assayed. From the results of such experiments (Fig. 5), it is obvious that SP2 transfection peaks earlier than that of SP82. Thus, when SP2 transfection is measured at the time of maximal SP82 transfection, the SP2 transfection level is depressed. The SP2 data in Fig. 1 are, so to speak, measurements taken at the "wrong" time. DISCUSSION The heuristic model underlying the experiments presented herein characterizes growth of cells in precompetency media as making the cells, in essence, diploid. To be able to incorporate extra DNA, these cells have to make room for the incoming DNA, and they accomplish this by halting DNA synthesis for one cell division. This is achieved for B. subtilis by the amino acid step down involved in diluting precompetency cells I{' TIME OF DNA ADDITION (MINUTES) FIG. 5. Twenty-minute pulses of transfecting DNA were given to cells at various times after precompetent cells were diluted 1:1 into competency medium. A l-,ig amount of SP2 DNA or SP82 DNA was added to 1 ml of cells, and 2 4.g of deoxyribonuclease was added 2 min later. The transfected cells were incubated for a total of 5 miii before being diluted and plated for plaque formation. into competency medium. Among the missing amino acids are those associaaed with cell wall synthesis. The result of these metabolic derangements is that there is an ability to fix DNA and a place to put it after entry. If the incoming DNA is an intact cell molecule, it can go to a free replicating site. If the DNA is not intact, the cells can incorporate some of the DNA by the process known as transformation. As this incorporated DNA is homologous to host DNA, it cannot immediately be replicated because the arrest of DNA synthesis was accomplished by sequestering needed enzymes. Phages bringing in heterologous DNA can effect synthesis of the needed enzymes if the DNA is that of the large virulent phages such as SP82. Two explanations of the low SP2 transfection were put forth: poor growth on competent cells and lysogenization of the host cells. The evidence of the studies is that there is little, if any, lysogenization, and that there is a sequestering of DNA synthesis enzymes. Although the present work has focused on DNA polymerase as the likely missing enzyme, the work of Kammen et al. (1) makes it likely that the ligase (6; Weiss '1lo

6 318 EPSTEIN BACTERIOL. REV. et al., Federation Proc., 26:395, 1967.) is also not functioning, thus leading to the possibility of a coordinated repression of both enzymes. The high recombination frequencies for SP82 transfection crosses can be accounted for by the high nuclease levels indicated by the experiments in Fig. 4. The data in Fig. 4 also permit the inference that replicating sites are involved in competency because the rise in competency for SP82 DNA and SP2 DNA is the same through about 6 min. Thus, there seems to be a common factor which would likely be one associated with cell properties because the phages are so different from each other. One obvious cell property is the number of replicating sites. Support for this interpretation has been obtained by measuring the UV capacity of competent cells. The capacity of 9-min competent cells is several times that of -min competent cells. It should also be noted that Table 1 showed a 1% efficiency of plating SP2 phages on 6-min competent cells. Therefore, the "true" transfection level for SPO2 DNA could be 1 times higher than that found, or close to 15/ml. This value is similar to that for SP82 transfection, indicating that it is the enzyme sequestering which is responsible for the low SP2 transfection level. Combining the results of Fig. 4 and 5 permits the conclusion that the time for maximal transfection for SP82 would be less than 9 min, if measured with.2,ug of DNA per ml, compared with 9 min when measured with 2,ug/ml. The net result depends on DNA fixation, amount of DNA available to recombine, and levels of nucleases. Thus, the term "competency" appears to be too general since its expression depends on at least one factor extraneous to the bacterium itself. The experiments with intact phages were presumed to involve attachment to representative cells in the competent culture, of which only about.1 % are actually transfectable. As shown in Table 2, this presumption has been tested by measuring the efficiency of plating of SPO2 phages at high multiplicities; similar low efficiencies were obtained. Thus, the enzyme sequestering must be occurring in all cells in the culture, not only in the transfectable ones. The most likely reason for the inability of the vast majority of the cells to be transfected would be that DNA fixation and enzyme sequestering are not necessarily coupled. However, it is possible that DNA fixation is maximal at all times in the competency medium and that nuclease levels are insufficiently reduced in most cells even when DNA synthesis enzymes have already been sequestered. Finally, the results of preliminary experiments on the DNA per cell support the model set forth at the beginning of this discussion. The DNA per colony-forming unit in - to 3-min competent cells is about twice that for 9-min competent cells. The difficulty in these measurements is the making of an accurate determination of the number of cells in each colony-forming unit. Length distributions have been determined from micrographs, and there is no change in the average length, which corresponds to about one and one-half of the smallest units measured. Still, no conclusion can be drawn about the DNA per cell until the length of the individual cells has been determined. The data of Wolstenholme et al. (19) permit the conclusion that broth-grown cells and 9-min competent cells have similar dimensions, but there is no information about the dimensions of the -min cells. [Since the presentation of this paper, R. N. Singh and M. P. Pitale (J. Bacteriol., 95: , 1968) have reported the existence of uninucleate and binucleate populations in competent cultures of B. subtilis, the uninucleate cells most likely being the ones actually competent.] ACKNOWLEDGMENTS This investigation was supported by grant GB 4497 from the National Science Foundation. I am indebted to Marilyn Flynn for excellent technical assistance. LITERATURE CITED 1. Aposhian, H. V., and A. Kornberg Enzymatic synthesis of deoxyribonucleic acid. IX. The polymerase formed after T2 bacteriophage infection of Escherichia coli: a new enzyme. J. Biol. Chem. 237: Doermann, A. H The vegetative state in the life cycle of bacteriophage: evidence for its occurrence and its genetic characterization. Cold Spring Harbor Symp. Quant. Biol. 18: Ephrussi-Taylor, H., and B. A. Freed Incorporation of thymidine and amino acids into deoxyribonucleic acid and acid-insoluble cell structures in pneumococcal cultures synchronized for competence to transform. J. Bacteriol. 87: Epstein, H. T Transfection enhancement by ultraviolet light. Biochem. Biophys. Res. Commun. 27: Epstein, H. T., and I. Mahler Mechanisms of enhancement of SP82 transfection. J. Virol. 2: Gefter, M. L., A. Becker, and J. Hurwitz The enzymatic repair of DNA. 1. Formation of circular X DNA. Proc. Natl. Acad. Sci. U.S. 58: Green, D. M Infectivity of DNA isolated from Bacillus subtilis bacteriophage, SP82. J. Mol. Biol. 1: Green, D. M Intracellular inactivation of infective SP82 bacteriophage DNA. J. Mol. Biol. 22:1-14.

7 VOL. 32, 1968 FACTORS AFFECTING BACTERIAL COMPETENCE Green, D. M Physical and genetic characterization of sheared infective SP82 bacteriophage DNA. J. Mol. Biol. 22: Kammen, H. O., R. J. Wojnar, and E. S. Canellakis Transformation in Bacillus subtilis. II. The development and maintenance of the competent state. Biochim. Biophys. Acta 123: Lerman, L. S., and L. J. Tolmach Genetic transformation. I. Cellular incorporation of DNA accompanying transformation in pneumococcus. Biochim. Biophys. Acta 26: Levinthal, C., and N. Visconti Growth and recombination in bacterial viruses. Genetics 38: McCarthy, C., and E. W. Nester Macromolecular synthesis in newly transformed cells of Bacillus subtilis. J. Bacteriol. 94: Mosig, G The effect of multiplicity of infection on recombination values in bacteriophage T4D. Z. Vererbungslehre 93: Okubo, S., and W. R. Romig Comparison of ultraviolet sensitivity of Bacillus subtilis bacteriophage SP2 and its infectious DNA. J. Mol. Biol. 14: Okubo, S., and W. R. Romig Impaired transformability of Bacillus subtilis mutant sensitive to mitomycin C and ultraviolet radiation. J. Mol. Biol. 15: Okubo, S., B. Strauss, and M. Stodolsky The possible role of recombination in the infection of competent Bacillus subtilis by bacteriophage deoxyribonucleic acid. Virology 24: Reilly, B. E., and J. Spizizen Bacteriophage deoxyribonucleate infection of competent Bacillus subtilis. J. Bacteriol. 89: Wolstenholme, D. R., C. A. Verneulen, and G. Venema Evidence for the involvement of membranous bodies in the processes leading to genetic transformation in Bacillus subtilis. J. Bacteriol. 92: