Genetic Modifiers of the Phenotypic Level of Deoxyribonucleic Acid-Conferred Novobiocin Resistance in Haemophilus

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov., 1966 Vol. 92, NO American Society for Microbiology Printed in U.S.A. Genetic Modifiers of the Phenotypic Level of Deoxyribonucleic Acid-Conferred Novobiocin Resistance in Haemophilus GRACE LEIDY, IRIS JAFFEE, AND HATTIE E. ALEXANDER Babies Hospital and Department of Pediatrics, Columbia University, College of Physicians and Surgeons, New York, New York Received for publication 11 August 1966 ABSTRACT LEIDY, GRACE (Columbia University, New York, N.Y.), IRIS JAFFEE, AND HATrrE E. ALEXANDER. Genetic modifiers of the phenotypic level of deoxyribonucleic acidconferred novobiocin resistance in Haemophilus. J. Bacteriol. 92: An apparent increase in novobiocin resistance in Haemophilus aegyptius after a second exposure to a particular H. influenzae transforming deoxyribonucleic acid was shown to be the result not of multi-step transformation but of the action of a gene functioning as an enhancement modifier. The modifier is very closely linked to a streptomycin resistance gene (which is linked to a novobiocin resistance marker); it affects the natural degree of resistance to both novobiocin and kanamycin to a measurable degree. Evidence of a repressor of the enhancement modifier is reported. We have reported (3) that both a novobiocin resistance transforming deoxyribonucleic acid (DNA) of Haemophilus influenzae strain Rd, resistant to 5,ug/ml of novobiocin, and of H. aegyptius 15, resistant to 0.4,ug/ml, conferred comparable levels of resistance in wild-type strains of both "species." Each DNA conferred resistance to 3 to 5,ug/ml of novobiocin in H. influenzae populations, but a much lower level (approximately 0.4,g/ml) in H. aegyptius populations. This difference in the level of DNA-conferred resistance was correlated with a "species" difference in sensitivity to novobiocin which distinguished those members examined. Wildtype H. aegyptius populations were approximately 5- to 10-fold more sensitive than H. influenzae populations. We proposed that these novobiocin resistance markers represented a similar singlestep spontaneous mutation in both "species." This hypothesis implied that a higher level of novobiocin resistance could not be conferred on H. aegyptius in multi-step transformations with the H. influenzae DNA. Our interest was therefore directed toward exploring the question of whether the H. influenzae novobiocin resistance gene might be qualitatively different and confer a higher level of resistance on H. aegyptius in multi-step transformations. Those results suggestive of multi-step transformations to a higher level of novobiocin resistance in H. aegyptius were consistent with the premise of an enhancement modifier of novobiocin resistance in a particular H. influenzae transforming DNA. Modifier (enhancement) genes which influence the level of streptomycin resistance conferred in pneumococcal transformations were first reported by Bryan (1). Evidence of an enhancement modifier and of a repressor of the modifier in Haemophilus is presented. MATERIALS AND MErHODS Strain designations. The symbol str-r indicates resistance to at least 1,000,ug/ml of streptomycin; nov-r, novobiocin-resistant; numeral following refers to level of novobiocin resistance. Recipient population. Wild-type H. aegyptius strain 15 (3) was used. DNA donors. Three independent spontaneous mutants of H. influenzae strain Rd were used, which were resistant to 5 jag/ml of novobiocin and at least 1,000,ug/ml of streptomycin, and were designated Rd str-r nov-r 5 (a), (b), or (c), respectively. DNA of Rd str-r nov-r 5 (a) is also referred to as "exceptional" DNA. Origin of novobiocin and streptomycin markers is indicated for H. aegyptius strain 15. The growth media, as well as the methods used to prepare competent cells and to measure transformants, have been reported (3). The "pour plate" rather than the "agar overlay" method of measuring transformants was used (3). 1464

2 VOL. 92, 1966 NOVOBIOCIN RESISTANCE IN HAEMOPHILUS 1465 RESULTS We wished to determine whether Rd H. influenzae transforming DNA from one-step spontaneous mutants resistant to 5,ug/mI of novobiocin could confer resistance to greater than 0.4,ug/ml on wild-type H. aegyptius by multi-step transformations. Our data, derived from extensive investigations, support the following conclusions. There was no unequivocal evidence that DNA of H. influenzae Rd resistant to 5,ug/ml of novobiocin could confer in H. aegyptius 15, by stepwise transformations, a degree of novobiocin resistance greater than 0.4,ug/ml. The results support our earlier (3) hypothesis that spontaneous mutation to 0.4,ug/ml of novobiocin in H. aegyptius 15 and to 5,ug/ml in H. influenzae Rd represents a comparable mutational event at a novobiocin locus. There is a relationship between the degree of sensitivity to novobiocin of wild-type strains of the two "species" and the level of novobiocin resistance conferred by the heterologous "species" DNA (3). An "exceptional" H. influenzae transforming DNA [DNA Rd str-r nov-r (a)], bearing the 5,ug/ml of novobiocin resistance marker as well as a linked streptomycin resistance marker, yielded results suggestive of multi-step transformation. Further study revealed, however, that wild-type H. aegyptius, first transformed only to resistance to 0.4 Jug/ml of novobiocin and then to streptomycin resistance by this same DNA, was rendered more resistant to novobiocin. Of the streptomycin-resistant transformants, 95% or more were resistant to 1,ug/ml of novobiocin. Experiments were therefore designed to determine TABLE 1. whether this transforming DNA was "exceptional," in that it contained a modifier (enhancer) of novobiocin resistance which was more closely linked to the streptomycin resistance marker than was the novobiocin resistance marker. Evidence of a novobiocin enhancement modifier in H. influenzae DNA [Rd str-r nov-r 5 (a)] revealed in wild-type H. aegyptius 15 populations. If an enhancement modifier of novobiocin resistance was intimately associated with the streptomycin resistance marker in the "exceptional" DNA Rd str-r nov-r 5, then, in wildtype H. aegyptius, transformants exhibiting the linkage relationship of the two markers should be more resistant to novobiocin than those transformed to novobiocin resistance only. To test this hypothesis, the activity of the "exceptional" H. influenzae transforming DNA in wild-type H. aegyptius was compared with that of two independent spontaneous mutant populations of Rd H. influenzae carrying the same two genetic markers, i.e., resistance to 5,ug/ml and at least 1,000,ig/ml of novobiocin and streptomycin, respectively. These latter two DNA preparations are designated Rd str-r nov-r 5 (b) and (c), and the "exceptional" DNA, Rd str-r nov-r 5 (a). H. aegyptius 15 DNA str-r nov-r 0.4 was included for comparison. The results of exposing competent wild-type H. aegyptius to these transforming DNA preparations are given in Table 1. The data show that the first H. influenzae DNA listed is "exceptional" in that (i) a significant number of transformants grew in the presence of 1,ug/ml novobiocin, virtually all of them resistant to streptomycin, and (ii) transformants which grew at the concentrations of the combined Evidence of modifier gene in a Haemophilus influenzae DNA which enhances novobiocin resistance of linked transformants in wild-type H. aegyptius DNA donor 0.2pg/mi Novobiocin 1,ug/mi Transformants per ml after incubation for 3 daysa Streptomycin Novobiocin (0.2 pg/ml) + Novobiocin (1 lg/ml) + (10 pg/mi) streptomycin (10 pg/ml) streptomycin (10,ug/ml) H. influenzae Rd str-r nov-r 5 (a) (29)b c 14 (24) str-r nov-r 5 (b) c 0 str-r nov-r 5 (c) (1) 111 H. aegyptius 15 str-r nov-r (3) c 0 a Figures in parentheses indicate the number of colonies after 6 days of incubation. b Of 20 colonies examined, 19 were resistant to streptomycin. c DNA Rd (a): 40 of 40 colonies were resistant to 1 ug/ml of novobiocin; DNA Rd (b): 19 of 20 colonies were sensitive to 1 ug/ml of novobiocin; DNA H. aegyptius: 19 of 20 colonies were sensitive to 1 jg/ml of novobiocin.

3 1466 LEIDY, JAFFEE, AND ALEXANDER J. BAcrERIOL. antibiotics used to select linked transformants (0.2 Ag/ml of novobiocin, 10 Ag/ml of streptomycin) were resistant to 1,ug/ml of novobiocin. These results indicate that linked transformants grew at a higher concentration of novobiocin than transformnants resistant to novobiocin alone. Confirmation of these results was obtained by isolating transformants which grew at concentrations of 0.2 Ag/ml of novobiocin or 10 Ag/ml of streptomycin after exposure to the "exceptional" DNA, and examining them for growth in the presence of 1,ug/ml of novobiocin and 10 or 100,ug/ml of streptomycin. Only those transformants in which both markers were simultaneously incorporated (linked) grew in the presence of 1 jig/ml of novobiocin. This was not true for the other DNA preparations (Table 1); linked transformants grew at a concentration of 0.2,Ag/ml of novobiocin, but less than 5% showed significant growth at a concentration of 1,g/ml. It appears, therefore, that, with the "exceptional" DNA as transforming DNA, novobiocin resistance is enhanced in H. aegyptius 15 when there is simultaneous incorporation of both markers. A search was made for linked transformants resistant to 0.2,ug/ml but not 1,ug/ml of novobiocin; of a total of 200 colonies examined in different experiments, none proved to be of this type. We noted that the growth rate of the linked transformants in medium which lacked streptomycin was generally lower than in medium which contained it. The enhancement phenomenon was not noted when strain Rd H. influenzae was used as receptor. A limited study with the "exceptional" DNA suggests that H. influenzae linked transformants may be slightly more sensitive to novobiocin than transformants resistant to novobiocin alone, and that the growth rate on agar lacking streptomycin may be slightly reduced. The modifier which enhances novobiocin resistance in H. aegyptius is not limited to H. influenzae. We subsequently obtained DNA from a spontaneous mutant population of H. aegyptius 15, resistant to 1,000,ug/ml of streptomycin and 0.4 Ag/ml of novobiocin, which acted similarly to DNA Rd (a) in the wild-type H. aegyptius population; linked transformants grew at a concentration of 1,ug/ml of novobiocin. Further evidence ofclose association ofmodifier with the streptomycin resistance marker of DNA Rd str-r nov-r S (a). The transforming activities of five different DNA preparations derived from H. aegyptius 15 resistant to streptomycin and novobiocin were compared in the wild-type H. aegyptius strain. The DNA- preparations varied only in the method by which resistance was attained by the strain from which they were isolated: by selection of spontaneous mutants or by transformation with the "exceptional" H. influenzae DNA [Rd str-r nov-r 5 (a)]. The data of Table 2 show that with one exception the transforming activities of the DNA preparations were similar to that of the "exceptional" H. influenzae DNA; i.e., a significant proportion of transformants grew in the presence of 1 or 1.5,ug/ml of novobiocin with incubation up to 6 days. Virtually all of these were streptomycin-resistant, indicating that linked transformants were selected at the higher novobiocin concentrations. The exception was the DNA donor in which novobiocin resistance was of transformation origin, but streptomycin resistance was of spontaneous mutational origin; linked transformants were sensitive to 1,ug/ml of novobiocin. These data support the inference that the "exceptional" H. influenzae DNA contains a modifier (enhancer) of novobiocin resistance which is more closely linked to the streptomycin marker than to the novobiocin resistance marker. Presence of a repressor in some transforming DNA preparations inhibiting the enhancement modifier. It is apparent from the DNA donors listed in Table 2 that two types of novobiocinresistant transformants were obtained after reexposure of streptomycin-resistant transformants to the "exceptional" H. influenzae DNA. One was resistant to 0.4,ug/ml of novobiocin, but sensitive to 1,ug/ml (donor I); the other was resistant to 1,ug/ml (donor V). It is also apparent from the data that certain of the DNA donors contain the modifier even though it was not expressed in the donor populations. The first three DNA donors listed have the modifier, because their DNA preparations acted similarly to DNA Rd str-r nov-r 5 (a). Greater than 99% of the DNA donor populations themselves, however, were sensitive to 1,ug/ml of novobiocin. These results suggest that there is an independent factor in these populations, also linked to the streptomycin resistance locus, which represses the action of the enhancement modifier. Two types of streptomycin-resistant transformants were therefore proposed to explain these differences. Presumably the last DNA donor listed does not contain the "repressor," since 100% of the population grew in the presence of 1,ug/ml. Failure to separate enhancement modifier from the streptomycin resistance marker of DNA Rd str-r nov-r 5 (a). Very few (generally less than 1 %) of transformants which grew in the presence of 1 Ag/ml of novobiocin after exposure of wild-type

4 VOL. 92, 1966 NOVOBIOCIN RESISTANCE IN HAEMOPHILUS 1467 TABLE 2. Influence of origin of linked markers in Haemophilus aegyptius DNA preparations on novobiocinresistant transformants growing at I,ug/ml of novobiocin, with wild-type H. aegyptius 15 as receptora H. aegyptius 15 DNA donorb Transformants per ml (5 to 6 days of incubation) DNA no. Novobiocin (jg/ml) Streptomycin 1st marker 2nd marker str-rtr nov-r 0.4'r I b nov-r 0.48p II b nov-r 0.4tr str-rlr III b str-rsp IV 1,240 1, , 320 str_rlr nov-r ltr V c 340 Symbols: tr = transformation origin, DNA Rd str-r nov-r 5 (a) only used; sp = spontaneous mutational origin. b Genetic markers in sequential order of origin. c Of colonies formed at 1 or 1.5,ug/ml of novobiocin 95% or more were resistant to streptomycin (20 to 40 colonies/dna sampled). H. aegyptius 15 to DNA Rd str-r nov-r 5 (a) were sensitive to streptomycin. One such transformant was selected for further study, because the possibility held that the enhancement modifier was independent of both the streptomycin resistance marker and the presumed repressor in this population. The transformant isolated was resistant to 3 Ag/ml of novobiocin and was used to select a streptomycin resistance marker of spontaneous mutational origin and transformation origin [DNA str-r nov-r 5 (a) as transforming DNA]. Transforming DNA preparations were derived from these cultures, and their activities in wildtype H. aegyptius 15 populations were examined. Results similar to those shown in Table 2 were obtained: virtually all linked transformants were sensitive to 1,ug/ml of novobiocin when the streptomycin resistance marker was of spontaneous mutational origin, but resistant if its origin was transformation by the "exceptional" H. influenzae DNA. The enhancement modifier could not be separated from the streptomycin resistance marker by this method. Sensitivity to novobiocin and kanamycin of streptomycin-resistant transformants containing the enhancement modifier with or without the repressor. Another approach was used to examine the effect of the enhancement modifier in H. aegyptius, because we were unable to separate it from the streptomycin marker. The sensitivity to novobiocin and kanamycin of cells transformed only to streptomycin resistance by the "exceptional" H. influenzae DNA was examined. As implied from the data of Table 2 and subsequently confirmed, two types of streptomycinresistant transformants were isolated after exposure of wild-type H. aegyptius to this DNA. One contained the enhancement modifier and its "repressor"; the other, the modifier without the repressor. The two types, each designated H. aegyptius 15 str-r en, were examined for their degree of sensitivity to novobiocin and kanamycin by the pour plate method (Table 3). Kanamycin was selected as another antibiotic, because a kanamycin resistance locus is linked to that of novobiocin and streptomycin in H. influenzae (2). Wild-type H. aegyptius and a streptomycinresistant transformant of DNA Rd str-r nov-r 5 (c) (Table 1) origin were included as controls. The lowest concentrations of kanamycin which were inhibitory (or partially inhibitory) for the wild-type strain were used. The data show that 15 str-r en ("repressed") was much more sensitive to 0.025,ug/ml of novobiocin than the "unrepressed" population which contained the enhancer. Presence of the enhancement modifier endowed the cells with greater resistance to kanamycin, but the repressor was not expressed. These differences were confirmed by the streak plate method (approximately 50 and 50,000 colony-forming units seeded). All of the streptomycin-resistant populations were resistant to 20,000,ug/ml of streptomycin. The presence or absence of the repressor in streptomycin-resistant transformants of DNA Rd str-r nov-r 5 (a) origin also influences the level of novobiocin resistance conferred by this DNA. Only a low proportion (<1%) of novobiocin-resistant transformants grew in the pres-

5 1468 LEIDY, JAFFEE, AND ALEXANDER J. BACTERIOL. TABLE 3. Influence of enhancement modifier and the repressor on sensitivity to novobiocin and kanamycina Per cent of colony-forming units with different amt (jag/ml) of: Haemophilus aegyptius 15 populations tested for sensitivity Novobiocin Kanamycin Wild-type str-r entr b (re pressed) str-r enir b (not re pressed) str-rer C a 120 to 250 colony-forming units seeded in pour plate preparations. b Streptomycin resistance of DNA Rd str-r nov-r 5 (a) origin. c Streptomycin resistance of DNA Rd str-r nov-r 5 (c) origin (Table 1). ence of 1 mg/ml of novobiocin when the "repressed" population was used as receptor. A much higher proportion (approximatey 70 to 85% of the number of transformants at 0.2,ug/ml) grew at a concentration of 1 gg/ml with incubation up to 6 days when "unrepressed" populations were used as receptors. DISCussIoN Bryan (1) has reported that, in pneumococci, a modifier (enhancement) gene, unlinked to a streptomycin resistance marker, affects the level of streptomycin resistance conferred by transforming DNA. The modifier alone did not confer any resistance to the cell. Rotheim and Ravin (5) reported that the enhancer is allele-specific. Modifier genes which influence the level of DNAconferred streptomycin resistance may differentiate the pneumococcus and streptococcus to a certain extent, as shown by Ravin and De Sa (4) Ẇe have presented evidence that a modifier gene, more closely linked to the streptomycin locus than the novobiocin resistance locus in H. influenzae and H. aegyptius, acts as an enhancer of novobiocin resistance in H. aegyptius. The modifier conceivably represents a mutation that occurred simultaneously with mutation to streptomycin resistance. Its activity is not specific for novobiocin, since the data of Table 3 suggest that it affects the degree of sensitivity to kanamycin as well as novobiocin. Further study may reveal a "species" difference in the type of action of the modifier; a limited study tentatively suggests that, in H. influenzae, cells transformed simultaneously to both novobiocin and streptomycin resistance by the "exceptional" H. influenzae DNA are slightly more sensitive to novobiocin than transformants resistant to novobiocin only. The repressor of the modifier is assumed to be less closely linked to the streptomycin marker than the enhancement modifier, since the two types of streptomycin-resistant transformants of H. aegyptius were demonstrated. The "species" origin of the repressor is not known. It will be of interest to explore the activity and specificity of the modifier and the repressor in other "species" of Haemophilus and to examine their influence on resistance to other antibiotics. ACKNOWLEDGMENT This investigation was supported by Public Health Service grants E 124 C-12, A Al, and GM from the National Institute of Allergy and Infectious Diseases and the National Institute of General Medical Sciences. LITERATURE CITED 1. BRYAN, B. E Genetic modifiers of streptomycin resistance in pneumococcus. J. Bacteriol. 82: GooDGAL, S. H Studies on transformation in Hemophilus influenzae. IV. Linked and unlinked transformations. J. Gen. Physiol. 45: LEIDY, G., I. JAFFEE, AND H. E. ALEXANDER Further evidence of a high degree of genetic homology between H. influenzae and H. aegyptius. Proc. Soc. Exptl. Biol. Med. 118: RAVIN, A. W., AND J. D. H. DE SA Genetic linkage of mutational sites affecting similar characters in pneumococcus and streptococcus. J. Bacteriol. 87: ROTHEIM, M. B., AND A. W. RAVIN The mapping of genetic loci affecting streptomycin resistance in pneumococcus. Genetics 46: