Marker Discrimination in Deoxyribonucleic Acid-Mediated ...

JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 608-618 Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 2 Printed in U.S.A.

Marker Discrimination in Deoxyribonucleic Acid-Mediated Transformation of Various Pneumococcus Strains GARARD TIRABY,I MAURICE S. FOX,* AND HARRIET BERNHEIMER Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,* and Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203 Received for publication 25 September 1974

The function responsible for discrimination among markers (point mutations) in Pneumococcus (hex) was traced back to the early strains used to demonstrate the chemical basis of transformation in the early 1940s. Those currently used laboratory strains failing to manifest this function arose from a single subline of the original strain. The function was also evident in other independently isolated strains including a number of different serological types. The hex function was not evident in transformation between heterologous pneumococcal strains probably as a result of the sensitivity of the function to saturation in the presence of deoxyribonucleic acid from closely related but nonisogenic strains.

Genetic markers used in pneumococcal transformation differ in the characteristic efficiency with which they are successfully integrated into the recipient genome. According to their individual integration efficiencies, markers fall into two principal classes, one with a high efficiency of integration (HE markers), the other with a 6to 10-fold reduced efficiency (LE markers) (8, 13, 23, 29). Although investigations concerned with marker effects in Pneumococcus were carried out in different laboratories, they were all performed with related noncapsulated strains derived from the same ancestor, strain D39S, an encapsulated, type II strain. Previous studies have shown that the bacterial function hex is responsible for these marker effects and acts by eliminating a large fraction of the LE transformants. Pneumococcal mutants deficient in the hex function have been discovered in stocks from sublines of rough variants of strain D39S or have been isolated after extensive mutagenesis (14, 16, 26, 28). hex mutants no longer discriminate between markers and integrate all markers with the same high efficiency. In the present study various strains derived from strain D39S were rendered competent and the integration efficiencies of well established LE and HE markers were measured to determine the phenotype of the strains with respect to the hex function. In addition, the behavior of several other pneumococcal strains was investigated. The latter strains were rough variants of

independent isolates of pneumococci of diverse capsular type. It was found that the various sublines derived from strain D39S display the hex+ phenotype with the exception of one branch in which the hex- phenotype was manifested, presumably as the result of a spontaneous mutation. The hex function appears to be a general property of Pneumococcus since all strains tested, although of diverse origin and serotype, discriminate between LE and HE mutations when transformed with homologous deoxyribonucleic acid (DNA). It was further found that the hex function exhibits little effect on markers carried by heterologous pneumococcal DNA. These observations suggest that the hex function is easily saturated, perhaps by the mismatched base pairs that are presumably present in the heteroduplexes formed in transformation of heterologous strains. While this work was in progress, similar observations on the saturation of the hex function by DNA from nonisogenic strains were described by Guild and Shoemaker at the Transformation Meeting, Tapoco, North Carolina (June 1973)

(9). MATERIALS AND METHODS Strains. Pneumococcal strains used as drug-sensitive recipients are listed in Table 1. They are all

rough variants of pneumococci of diverse origin. Strains derived from D39S, a type II strain, are designated by trivial names used in previous publications. Other strains, designated by their number in the col'Present address: Centre de Recherche de Biochimie et lection of one of us (H. Bernheimer) will sometimes be de Genetique Cellulaires, 118 Route de Narbonne, Toulouse, followed by a Roman numeral indicating the capsular France. type of the smooth strain from which they were de608

VOL. 121, 1975

MARKER EFFECTS I :N PNEUMOCOCCUS

TABLE 1. Transformable noncapsulated pneumococcal strains used

609

in 1.5 ml of 0.1% sodium deoxycholate dissolved in 0.15 M sodium chloride-0.015 M sodium citrate (SSC). The lysed cells were incubated with 250 gg of Pronase at 45 C until a clear viscous solution was obtained Capsular Strain Other of pa- References type strain a number number designation (about 30 min). desigationrent Streptococcal DNA was prepared by the same procedure used to obtain pneumococcal DNA after lysis II 654 D39R Austrian and of the cells by the method of Tiraby and Fox (25). Bemheimer, Bacillus subtilis DNA prepared by the procedure unpublished of Haseltine and Fox (11) was provided by S. Fornili. -4 II R36NC 18 Transformation. Cultures were initiated by ino5 R36A II 1 culating B medium with bacteria from frozen or lyR6 II 19 ophilized stocks. Cells were then grown in CH medium II R6x 26 for several generations. To obtain the highest level of II 6 Cl3 competence, cells had to be passed back and forth II 401 29 through the two media at least three times. DifferSIII-1 II 24 ences were observed between the strains in the growth Rx II 20 rate, in the final density of cells, or in the lag time 655 Lederle II Austrian and after freezing and thawing, but since these differences R Bernheimer, have no bearing on the results described in this report, unpublished the individual characteristics of each strain are not II 656 Rat R Austrain and described. Cells were finally made competent by the Bernheimer, procedure of Tiraby and Fox (27) or by the procedure unpublished of Bernheimer and Wermundsen (2). Freshly preI 477 2 pared or frozen (-70 C) competent cells were used. 662 A66R III 4 The fresh competent cells were exposed to DNA for 1018 VIR VI 4 20 min at 30 C and the culture was diluted five times VII 1013 VIIR 4 in B medium. Thawed competent cells were diluted VIII 264 VHIR- 1 18 five times in prewarmed competence medium, exaStrain number in personal collection of H. Bern- posed to DNA for 20 min at 30 C and then diluted with 1 volume of B medium. Where indicated, the dilution heimer. medium contained 10 Ag of deoxyribonuclease per ml (Worthington) and 0.001 M magnesium sulfate. After rived. The two strains 655 and 656 derived from type dilution, all cultures were incubated for 2 h at 37 C to II strains are unrelated to each other or to the D39S allow for phenotypic expression of transformants. sublines; the smooth parent of 655 was a human isolate Glycerol was added to a final concentration of 10% and the parent of 656 was isolated from a rat. prior to freezing the transformed cultures at -70 C. Pneumococcal DNA was extracted from strains in- Glycerol prevents the loss of viability of all cells indicated in the text and from strain 119, a derivative of cluding transformants, and no loss of transformants R6 carrying markers conferring resistance to sulfona- was observed after 6 months of storage. Thawing and mide (sulf-d), streptomycin (str-r41), fusidic acid (fus- refreezing of the transformed cultures can result in a rA), novobiocin (nov-rl), rifampin (rif-r-23), and opto- reduction in the number of transformants. To avoid this inconvenience, the cultures were divided into chin (opt-r2). Streptococcal DNA was extracted from strain several volumes prior to freezing, and each volume was used only once after thawing. Wicky, a group H streptococcus (25). Media. Cells were grown and maintained in B medUnless otherwise noted, transformants of all strains ium (21). The transformation medium (CH medium) were selected at the following drug concentrations: described by Gurney and Fox (10) was modified by re- sulfonamide, 80 ug/ml; streptomycin, 200 gg/ml; placing fresh yeast extract with yeast extract pre- fusidic acid, 50 Ag/ml; novobiocin, 4 ug/ml; streppared by the procedure of Sicard (22). When com- tolidigin, 5 gg/ml; rifampin, 2 zg/ml; optochin, petence was achieved by the procedure of Bernheimer 2 yg/ml. et al. (4), uridine, adenosine, and pyruvate were omitDNA shearing. The concentration of NaCl of a ted from the CH medium. Strains Cl and 401 re- solution of DNA, 10 Mg/ml in SSC, was made 1 M. A quired glutamine (200 ug/ml) for better growth in all 5-ml amount of the DNA solution was stirred at 4 C media. Transformants were selected in M medium in a fluted glass flask with a 1-cm diameter stainless (25) containing the selective agent and 1% citrated blade of a Virtis 45 homogenizer (Virtis Co., Gardiner, human or horse blood. N.Y.). The duration of stirring was 2 h at the maxiPreparation of DNA. Purified pneumococcal mum speed of the homogenizer, about 42,000 rpm. Antibiotics. Streptomycin sulfate USP was purDNA was prepared by the procedure of Rosenthal and Fox (21) from 2-liter cultures. Experiments involving chased from Eli Lilly and Co. Optochin and sulfanilaDNA from strain 119 were all carried out with the mide were purchased from Calbiochem. Streptolidigin same preparation of DNA. Pronase-treated lysates of was a gift of the Upjohn Co. Fusidic acid was a gift of cells possessing good transforming activity were pre- Leo Pharmaceutical Products, Inc., Ballerup, Denpared by growing 10-ml cultures to the stationary mark. Rifampin was a gift of the Ciba Pharmaceutical phase, spinning down the cells, and suspending them Co., N.J.

TIRABY, FOX, AND BERNHEIMER

610

Sulfanilamide (5 mg/ml in sterile water) was stored room temperature. Solutions in sterile water of streptomycin (200 mg/ml), optochin (2 mg/ml), and fusidic acid (10 mg/ml) were kept at 4 C. Rifampin and streptolidigin were first dissolved in dimethyl sulfoxide (50 mg/ml); the solutions were then diluted 10-fold in sterile water. Streptolidigin was stored at 20 C and rifampin was stored at 4 C.

at

-

RESULTS

Marker efficiencies in strains derived from strain D39S. Many pneumococcal strains used in transformation studies are derived from the same parent. The lineage of the most widely used strains is shown in Fig. 1. It was of interest to examine the response of these strains, when grown in the same medium and rendered competent by the same procedure, toward some widely used and some more recently described genetic markers with different characteristic integration efficiencies. The relative integration efficiencies of markers carried by the DNA of strain 119 (a derivative of R6) were therefore measured in these strains. The integration efficiency of a given marker is the ratio of the transformant yield for this marker to the transformant yield for a reference marker (15). The integration efficiencies of two HE and two LE markers on the various sublines of D39S are shown in Table 2. The level 139S

(1928)

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FIG. 1. Geneology of some Pneumococcus strains widely used in transformation studies. Dates in brackets correspond to the year of the first publication mentioning the strain in question. Strains not listed in Table 1 were isolated by Dawson (5), D39S; Avery et al. (1), R36; Taylor (24), SIII N. (a) Nitrosoguanidine-induced mutation (hex+--+ hex-) and introduced by transformation into strain Cl3.

(b) Spontaneous mutation (Smooth

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J. BACTrERIOL .

of discrimination of the rough variant D39R isolated about 15 years ago from the smooth strain D39S and preserved in the lyophilized 0tate is the same as that of strains R36A, R36NC, and R6. According to the criterion of absence of discrimination between these LE and HE markers, strains R6x, Rx, and S8111 are hex-. R6x was isolated after transformation of strain R6 by DNA from strain Rx (Fig. 1). Since it has been shown that a single mutation is sufficient to result in the loss of the capacity to discriminate (14, 17, 26), it would appear that a hex- mutation had occurred at the time of transformation of R36A by DNA from strain A66, or during the early propagation of the transformant strain. Presumably, strain SIII-N is also hex-, but since the strain was not available, it could not be tested. For the recipient strains listed in Table 2, essentially the same levels of discrimination were observed when either low or high concentrations of DNA were used for transformation (data not shown). It was found, however, that the integration efficiencies of the same markers on recipient strains Cl3 and 401 were dependent on the concentration of DNA 119 used (Table 3). Strain 401 is a mutant of Cl3 recognized by its reduced capacity to discriminate between HE and LE markers. As much as a threefold increase in efficiency was found for LE markers in strain Cl3 with saturating concentration of DNA. Strain 401 exhibits little variation in marker efficiency with DNA concentration. If strain Cl3 is transformed with DNA isolated from strain Cl3 carrying genetic markers, the transformation yield with respect to LE and HE markers is independent of DNA concentration (8; G. Tiraby, unpublished observations). The DNA concentration effect observed in transformation of strain Cl3 by DNA from strain 119 might be due to differences in nucleotide sequence in the genomes of R6 and Cl3. Although R6 and Cl3 are two sublines of the same parent, R36A, the two strains were used in different laboratories and cultivated in different media for more than two decades. At the present time, strain Cl3 differs from R6 or from the other D39S sublines in many respects: strain Cl3, which is generally more resistant to antibiotics than R6, is glutamine dependent but shikimic acid independent, whereas R6 is glutamine independent but shikimic acid dependent (G. Tiraby, unpublished observations). Furthermore, the hypothesis of variation in nucleotide sequence between Cl3 and R6 is supported by the finding that the marker fus-rA, high efficiency in R6, is low efficiency in C13. Discrimination by pneumococcal strains

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611

TABLE 2. Relative integration efficiencies of several markers determined on pneumococcal strains derived from the same ancestor" Markers

D39Rb Ac

Str-r41 Fus-rA Nov-rl Stg-rF

R36NC

R36A

Drug resistance

Streptomycin Fusidic acid Novobiocin Streptolydigin

430 360 40 45

Bd

B

A

A

B

R6 A

R6x B

A

SIR, B

1 3,000 1 260 1 2,750 1 685 1 0.84 3,400 1.1 280 1.1 3,350 1.2 760 1.1 0.09 295 0.10 20 0.08 280 0.1 0.10 215 0.07 16 0.06 265 0.1 820 1.2

Rx

A

B

A

B

335 355 360 370

1 1.1 1.1 1.1

475 450 470 430

1 0.9 1.0 0.9

a Cells grown in the competence medium to a density of about 106 colony-forming units per ml were diluted 10 times in prewarmed competence medium containing 1 sg of DNA per ml from strain 119. The 20-min incubation period at 30 C was followed by a further 2-h incubation period at 37 C. Thereafter, 10% glycerol was added, a portion of the culture was frozen at -70 C, and the remaining fraction was used to estimate the number of transformants. The following day a more accurate number of transformants for each was determined by plating appropriate dilutions of the thawed transformed culture. b Recipient strain. c Column A throughout = number of transformants x 108/ml. d Column B throughout = integration efficiency (ratio of the number of transformants for a given marker to the number of transformants for the str-r41 marker).

TABLE 3. Marker ratios on transformation of strains Cl, and 401 with nonsaturating and saturating concentrations of DNA from strain 119a Markers

Str-r41 Sulf-d Fus-rA Nov-rl Stg-rF

Antibiotic resistance

Streptomycin Sulfanilamide Fusidic acid Novobiocin Streptolydigin

Cl,

401

Ab

BCe

CCd

B

A

B

C

B

80 135 12 11.2 10.5

1 1.70 0.15 0.14 0.13

545 655 190 175 220

1 1.2 0.35 0.32 0.40

32.5 26 22 14.5 17

1 0.80 0.68 0.45 0.52

190 135 170 110 115

1 0.71 0.89 0.58 0.60

"Freshly prepared competent cells of strains Cl, and 401 were-exposed to DNA for 20 min at 30 C, and then diluted five times in deoxyribonuclease B medium. After 2 h of incubation at 37 C, 10% glycerol was added, and the cultures were frozen at -70 C. The number of transformants was determined by plating 1 day later the thawed cells in a selective medium. Column A throughout = number of transformants x 103/ml with nonsaturating concentration of DNA (0.02 sg/ml). c Column B throughout = integration efficiency (marker ratios). dColumn C throughout = number of transformants x 103/ml with saturating concentration of DNA (5 Mg/nml).

of diverse origin: markers carried by DNA from a heterologous strain. Various noncapsulated strains derived from strains of different serotypes were transformed with DNA from strain 119. Using saturating concentrations of DNA, all markers were integrated with about the same efficiency (Table 4). The effect of DNA concentration on the discrimination property of several strains was investigated. Figure 2 depicts the yield of transformants of recipient strain 1013 for various markers as a function of the DNA concentration. It can be seen (Fig. 2) that the marker ratios are nearly independent of the DNA concentration. Strains 662 and 264 behave similarly. On the basis of marker efficiency, all of these strains appear similar to the hex- strain. Exposure of DNA to ultraviolet (UV) light

results in a loss of the biological activity of the DNA. When tested on a hex+ strain, LE markers are much more sensitive to inactivation than are HE markers. The rate of inactivation of the markers str-r41 and nov-rl carried by DNA 119 treated with UV light was determined in several strains of apparent hex- phenotype and in the two reference strains R6 (hex+) and R6x (hex-) (Fig. 3). The two markers are inactivated at about the same rate in strains 264, 477, and 662 and at the rate corresponding to HE markers in the hex+ strain. According to this criterion, all the experimental strains tested manifest the hex- phenotype. This result was unexpected since it has been shown that strain R6x, a typical hex- strain, has lost a function by mutation, and the results described in Fig. 3 would suggest that the other independently

J. BACrERIOL.

TIRABY, FOX, AND BERNHEIMER

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