Homospecific DNA was obtained from the SIII-1 strain of pneumococcus, heterospecific DNA from the Challis strain of group H streptococcus. Since the Rx and ...
RECOGNITION O F STREPTOCOCCAL DNA BY A MUTANT PNEUMOCOCCUS UNABLE TO DISCRIMINATE AMONG MARKERS IN PNEUMOCOCCAL DNA GERARD TIRABY
AND
ARNOLD W. RAVIN
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and Department of Biology, Uniuersity of Chicago, Chicago, Illinois 60637 Manuscript received March 20, 1973 Transmitted by ROCHELLE E. ESPOSITO ABSTRACT
A mutant strain of pneumococcus which fails to discriminate against lowefficiency markers during transforniation by homospecific pneumococcal donor DNA retains the wild-type capacity to discriminate against heterospecific (streptococcal) donor DNA. W e conclude that discrimination against heterospecific DNA must differ from that against low-efficiency markers by the kind or number of elements being recognized.
ENETIC markers used in pneumococcal or streptococcal transformation differ in the efficiency with which they are integrated into the genome of a recipient bacterium, less efficient markers being designated as low-efficiency (LE) in contradistinction to the more efficient markers called high-efficiency (HE; SICARD and EPHRUSSI-TAYLOR 1965; LACKS 1966), Efficiency of integration does not depend solely upon the nature of the marker, but it is also affected by heterology in the donor DNA (SCHAEFFER 1958; CHENand RAVIN 1966). Thus, DNA from a related species (heterospecific DNA) is usually less efficient in transformation than is DNA from the same species (homospecific DNA). Some, although not all, of the effects of heterologous DNA on transformation can be detected after its integration into a recipient genome, indicating that neighboring nucleotide sequences affect marker integration (BISWAS and RAVIN1971; RAVINand MA 1972). The relative inefficiency of heterospecific DNA can be reduced by prior treatment of recipient bacteria by heat (RAVINand MA 1972) or by certain inhibitors of nucleic acid synthesis (DEDDISH and RAVIN 1972, and in preparation). It was observed that tramformation by specifically LE markers in the transformation of pneumococcus or streptococcus with homospecific DNA was also enhanced as a consequence of treating recipient cells with heat or inhibitors. Therefore it seemed possible that discrimination against LE markers and discrimination against heterospecific DNA might be due to a common mechanism. A way of testing this possibility involves the use of a class of mutants that has been discovered in certain transformable strains of pneumococcus. These mutants Genetics 7 4 : 557-562 August, 1973.
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G . TIRABY A N D A. W. R A V I N
fail to distinguish between LE and HE markers in homospecific DNA, both types of markers being integrated with relatively high efficiency (LACKS 1970; TIRABY 1971). One such strain, Rx, was originally isolated by one of us for its deficiency in capsule synthesis (RAVIN1959), and was subsequently shown to differ from another strain, Rz, in respect to its efficiency as a recipient in integrating a streptomycin-resistance ( s t r - r l )marker (GREEN1959). The Rx strain was found to be nondiscriminating with respect to an LE novobiocin resistance marker (PETERSON and GUILD1968) and, more recently, to a large number of LE markand Fox, in preparation). ers (TIRABY To determine whether discrimillation against heterospecific DNA is identical to that against LE markers, both types of DNA can be presented to the Rx strain as well as to strains that normally discriminate against LE markers. If transformation by heterospecific DNA were as efficient as homospecific in the Rx strain specifically, the mechanisms underlying the two forms of discrimination could be regarded as identical. The experiments described here were undertaken with this kind of test in mind. MATERIALS A N D METHODS
In one set of experiments the Rx strain was compared with the SIII-1 strain of pneumococcus. Both strains are closely-related, having descended from the unencapsulated R36A strain of Avery (RAVIN1959). In addition, the Challis strain of streptococcus was tested as a heterospecific control recipient. In these experiments recipient cells previously made competent and frozen i n the usual way (CHENand RAVIN1966) were thawed in icewater and 2 x IO7 cells were inoculated into 25. ml of NS medium containing 1 pg/ml of DNA, which had been isolated from an appropriately marked donor strain and purified in the usual manner (CHEN and RAVIN1966). This culture was incubated a t 30°C for 20 minutes. at which time exposure of cells to DNA was terminated by the addition of an excess of Mg-activated pancreatic deoxyribonuclease. The culture was then transferred to 37°C 2nd allowed to grow for 3 hours, at which time phenotypic expression of all donor antibiotic resistance markers was already complete. The cells were then plated on selective media to determine the number of bacteria transformed for each donor marker. The selective concentrations used were in every case below the maximum level tolerated by the transformed strain: 100 pg/ml streptomycin; 0.25 pg/ml erythromycin; 0.5 pg/ml lincomycin. Homospecific DNA was obtained from the SIII-1 strain of pneumococcus, heterospecific DNA from the Challis strain of group H streptococcus. Since the Rx and SIII-1 strains have had different histories, albEit a common ancestor, it was desirable to test two recipient strains differing in genotype only by the mutation shown to be responsible for the lack of discrimination in the Rx strain. This was accomplished by transferring this mutation (called h e r ) into the R6 strain, itself a derivative of R36A, by a procedure described in detail elsewhere (TIRABY and Fox, in preparation). The strain so produced, called E6x, was compared with its parent strain, R6. In this set of experiments transformation was carried out according t o procedures described by GURNEYand Fox (1968). These procedures were essentially similar to those described above, except that uptake of DNA was terminated by a four-fold dilution of the medium in which the cells were incubating. Plating was carried out after two and one-half hours of further incubation at 37"C, when phenotypic expression of all acquired donor markers was complete. The donor markers conferred resistance to various antibiotics and drugs. The concentrations used for selection were less than the maxima tolerated by the respective transformants: 200 pg/ml streptomycin; 5 pg/ml streptolydigin; 5 pg/ml rifampin; 50 pg/ml fusidic acid; 0.25 pg/ml erythromycin. Homospecific DNA was obtained from pneumococcal strains SIII-1 and R6, heterospecific DNA from the Wicky strain, a group H streptococcus similar to the Challis strain.
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DISCRIMINATION I N TRANSFORMATION
The genetic symbols employed are: str, ery, Zin, fus, stg and rif, which refer to loci controlling resistance to streptomycin, erythromycin, lincomycin, fusidic acid, streptolydigin and rifampin, respectively. The recipient strains Rx, SIII-1, K6, R6x, and Challis are sensitive to all of the antibiotics. Independently-obtained resistance alleles of the respective loci are designated 4 5 3 or -r2 or -rA, etc. R E S U L T S A N D DISCUSSION
In Table 1 it may be seen that the pneumococcal markers str-r53 and ery-r2 have equal efficiencies in transforming strains SIII-1 and Rx. However, while Zin-r2 has about one-tenth the efficiency of str-r49 when transforming SIII-1, the two markers have about the same efficiency when transforming the Rx strain. Despite the failure of Rx to discriminate against Zin-r2, an LE marker in transforming SIII-1, Rx discriminates against heterospecific Challis DNA as much as SIII-1 does. Yet the Challis DNA is not intrinsically of low biological activity, since it transforms Challis cells about 25 times more efficiently than does SIII-1 DNA str-r53 ery-r2. The latter DNA, on the contrary, transforms the SIII-1 strain about 30 times more efficiently than does Challis DNA. The results shown in Table 1 are typical of those obtained in several similar experiments. Therefore, it may be concluded that the mutation that renders Rx incapable of discriminating against the LE marker Zin-r2 does not cause a similar lack of discrimination against heterospecific DNA. This conclusion is reinforced by the observations obtained by comparing isogenic strains R6 and R6x which differ only by the hex- mutation contained in Rx. Table 2 shows results typical of those obtained in repeated experiments. It may be seen that R6x fails to discriminate against markers stg-rF and rif-r27, which behave as LE markers in transforming TABLE 1 Comparison of pneumococcal strains S I I I - l and R x with respect io discrimination against LE markers and heteraspecific DNA Relative efficiency 01 markers
No. of transformants X 104,”l Recipient
Donor
Nature of cross
pneumo SIII-1
(1) str-r53 ery-r:! (2) str-r49 lin-rl (3) str-r43 ery-ri4 (1) str-r53 ery-rz (2) str-r49 lin-rl (3) str-r43 ery-rl4 (3) str-r43 ery-rl4 ( 1 ) str-r53 ery-r2
Homo Homo Het Homo Homo Het Homo Het
pneumo Rx strepto Challis
sfr
33.4 20.0 1.1 43.0 17.0 0.9 68.2 2.4
erv
29.4 ...
1.1 51.5
lin
c’q’
,str
.
0.88
2.8
. .
,
. .
.. ..
. . .
14.3
1.1 33.2 2.6
. ... . . .
1.o 1.1 1.2 0.50 0.92
of het. transf.
___
lin
-
het. str. ~
str
homo. str.
..
... ...
0.14
0.03 ...
...
0.84
..
... ... . .
0.02 ...
0.04
The donors of DNA preparations (I), (2), and (3) are respectively SIII-1 se-r53 ery-r2, SIII-1 str-r49 lin-rl and Challis str-r43 ery-rl4. The various markers arose spontaneously in the indicated strains. The relative efficiency of the heterospecific transformation is the ratio of the number of str transformants obtained with DNA ( 3 ) to the number obtained with DNA (1) when the recipient is pneumococcus; when the recipient is streptococcus the ratio is the number obtained with DNA (1) to that obtained with DNA (3).
560
G. TIRABY A N D A. W. R A V I N W
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561
R6. Yet R6x discriminates against streptococcal Wicky DNA as well as does R6. Some additional observations may be made. The SIII-1 markers str-r53 and ery-r2 are of equal and high efficiency in transforming SIII-1 and R6x (Tables 1 and 2). Yet str-r53 has only about one-tenth the efficiency of ery-r2 in transforming R6 (Table 2). One possible explanation is that SIII-1 and R6 have genetically different patterns of marker discrimination. Another possibility is that, while the SIII-1 DNA (except for the str-r53 and ery-r2 markers themselves) is homologous with that of the SIII-1 recipient, it differs from that of R6 by the presence in the latter of a silent LE mutation linked to the site of the str-r53 mutation. In either case, however, it is clear that discriminating recipients of an originally identical percentage may evolve to differ in their efficiencies of transformation by specific markers. The pneumococcal HE marker str-r.ll transferred into Wicky recipients serves as a useful reference marker in comparing the efficiencies of Wicky markers (Table 2). Thus, it may be seen that the fus-rl and rif-7-2markers in Wicky DNA are equally efficient in transforming Wicky, but are decidedly unequal in efficiency of transforming R6. Yet the inefficiency of rif-r-2 (as well as stg-r10) relative to f u s - ~ lis maintained in transforming R6x. Thus, the R6x strain’s failure to discriminate among markers does not apply when the DNA is heterospecific. So far as the principal object of these experiments is concerned, however, we may conclude that discrimination against heterospecific DNA must differ from that against low-efficiencymarkers by the kind and/or number of elements being recognized. On the plausible assumption that recognition is due to the specificity of an enzyme acting uporr DNA, we are led to suppose either: (1) that a single enzymatic system, which recognizes both heterospecific DNA and LE markers, may be so altered by mutation as to have its effectiveness against the latter specifically reduced; or (2) that different enzymes are responsible for the recognition of heterospecific DNA and LE markers in homospecific DNA. The observation that physical or chemical agents can affect both forms of discrimination (RAVINand MA 1972; DEDDISH and RAVIN,in preparation) is compatible with either alternative: these agents may act upon a common enzymatic process or upon biochemical steps common and prior to distinct enzymatic precesses of discrimination. This research was supported by grants AI 09117-05 and AI 05388-10 from the National Institute of Allergy and Infectious Disease. G.T.was supported principally by a fellowship from the Pierre Philippe Foundation and a fellowship from the Foundation for Research in Medicine and Biology. Part of this work was done by G.T.in the laboratory of PROFESSOR MAURICES . Fox. The technical assistance of MR. MICHAELM A i n some of these experiments is gratefully acknowledged. LITERATURE CITED
BISWAS,G. D. and A. W. RAVIN,1971 Heterospecific transformation of pneumococcus and streptococcus. IV. Variations in hybrid DNA produced by recombination. Molec. Gen. Genetics 110: 1-22. CHEN,K. C. and A. W. RAVIN,1966 Heterospecific transformation of pneumococcus and streptococcus. I. Relative efficiency and specificity of helping effect. J. Mol. Biol. 22: 109-122.
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G . TIRABY AND A. W. RAVIN
DEDDISH,P. E. and A. W. RAVIN,1972 Relation of macromolecular synthesis to efficiency of streptococcal transformation by markers of homospecific and heterospecific origin. Abstr. Ann. Meeting Amer. Soc. Microbiol. 6122: 50. GREEN,D. McD., 1959 A host specific variation affecting relative frequency of two markers in pneumococcus. Exp. Cell Res. 18: 211-231. GURNEY,T. and M. S. Fox, 1968 Physical and genetic hybrids formed in bacterial transformation. J. Mol. Biol. 32: 83-100. LACKS,S., 1966 Integration efficiency and genetic recombination in pneumococcal transIormation. Genetics 53: 207-235. -, 1970 Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and activities possibly pertinent to genetic transformation. J. Bacteriol. 101 : 373-383.
J. M. and W. R. GUILD,1968 Fractionated strands of bacterial deoxyribonucleic PETERSON, acid. 111. Transformation efficiencies and rates of phenotypic expression. J. Bacteriol. 96 : 1991-1 996. RAVIN,A. W., 1959 Reciprocal capsular transformations of pneumococci. J. Bacteriol. 77: 296-309. RAVIN,A. W. and M. MA, 1972 Specific effects of heating of transformable streptococci on their ability to discriminate between homospecific, heterospecific and hybrid deoxyribonucleic acid. J. Bacteriol. 109: 616-625. SCHAEFFER, P., 1958 Interspecific reactions in bacterial transformations. Symp. Soc. Exp. Biol. 12: 60-74. 1965 Genetic recombination in DNA-induced transSICARD,A.-M. and H. EPHRUSSI-TAYLOR, formation of pneumococcus. 11. Mapping the ami-A region. Genetics 52 : 1207-1227.
TIRABY, G., 1971 Etude des mecanismes responsables des efficacites de transformation chez Diplococcus pneumoniae. These, Docteur de specialite, Universite Paul Sabatier, Toulouse.
