a transforming marker that produces …replicon by donor dna that has failed to become physically...

A TRANSFORMING MARKER THAT PRODUCES MERODIPLOIDS WITH HIGH EFFICIENCY AND STABLE TRANSFORMANTS

WITH LOW EFFICIENCY IN STREPTOCOCCUS ARNOLD W. RAVIN AND MICHAEL M A

Departments of Biology and Microbiology, Uniuersity of Chicago, Chicago, Illinois 60637

Manuscript received February 4, 1975

ABSTRACT

A mutation (ery-r8) conferring a high level of resistance to erythromycin in the Challis strain of Streptoccus sanguis can be transferred to wild-type erythromycin-sensitive recipients via single molecules of donor DNA. The transformants thus produced are of two types: (1) cells slightly more resistant to erythromycin than wild-type and capable of segregating (at a frequency of 2 X 10-*/bacterium/generation) either wild-type or highly-resistant cells like the original donor type; (2) cells phenotypically and genotypically identical to the original donor type. The unstable diploids ( e r y - r t / - t ) occur with a frequency equivalent to that obtained with high-efficiency (HE) markers, whereas the stable donosr-type (ery-r8) transformants occur with about five hundred times lower frequency. Penetration of the wild-type recipient by more than one molecule of DNA bearing the ery-r8 marker increases by as much as seven times the incidence of stable transformants. UV-irradiation of molecules bearing the ery-r8 marker diminishes their ability to cooperate in producing a stable transformant, although the UV sensitivity of stable transformant production by a single DNA molecule is not different from that of diploid production. Hence, stable transformants do not appear to be produced by a process typical of low efficiency (LE) markers, which are generally highly sensitive to ultraviolet irradiation. Moreover, stable ery-r8 transformants are produced with equally low frequencies in strains of S. pneumoniae that discriminate ( h e z f ) and fail to discriminate (hex-) between H E and LE markers. We postulate that all transformations by the ery-r8 marker result in e r y - r t / f diploids, and that segregation results in the infrequent stable transformants pf the original donor type. This hypothesis is supported by the observations that rifampin treatment of ery-r t /+ populations increases the frequency of segregation and similar treatment of wild-type recipients under- going transformation by the ery-rg marker increases the frequency of stable transformants.-In producing the ery-rt/+ transformant the r8 allele is integrated close to the site of its wild-type homolog, since single molecules d DNA from this transformant can be shown to carry both alleles. Segrega- tion of either the ery-r8 o r + allele is not detectably enhanced by acridine orange or thymidine deprivation.-The ery-r8 marker occurs close to a site of mutation (ery-r2) which confers erythromycin resistance upon ribosomes. When the r2 and r8 markers are jointly transferred, ery-r2-r8/+ genomes are produced in which the r2 marker is stably integrated but the r8 marker is unstably adjoined t o its wild-type homolog, Thus, the duplicated region can be quite short. When the ery-r8 marker is stably integrated, the region of the

Genetics 80: 421443 July, 1975.

422 A. W. RAVIN A N D M. M A

marker is refractory to subsequent transformation. Markers with properties like ery-r8 are not particularly rare, being found with a frequency of about 4% among spontaneous mutations to erythromycin resistance.

I N the transformation of recipient bacteria by DNA extracted from genetically marked strains, markers in the donor DNA usually replace their homologous

sites in the recipient genomes. Exceptionally a donor marker is added to the genome without concomitant loss of its allele (HOTCHKISS and ABE 1968; BERN- HEIMER and WERMUNDSEN 1969; RAVIN and TAKAHASHI 1970; AUDIT and ANAGNOSTOPOULOS 1973). The simultaneous presence of donor marker and recipient homolog is usually detected by evidence of transforming activity of both alleles in DNA extracted from such rare diploids (RAVIN and TAKAHASHI 1970). The rarity of diploidy resulting from transformation indicates the high precision of LLmatching’’ or “pairing” between recipient and donor genomes in the course of genetic recombination during transformation. Conversely, the‘ diploids produced by exceptional donor markers constitute a sign of imprecise pairing or “mismatching.”

A replicated diploid state could be due either to establishment of a separate replicon by donor DNA that has failed to become physically intergrated into the recipient genome or to an integration event that inserts into the recipient DNA a segment of the donor DNA that duplicates a corresponding recipient region in ail respects except for the marker itself.

We have discovered a marker that produces diploid transformants with high efficiency and substituted haploids with low efficiency. In an analysis of transformation by this marker, which confers a high level of resistance to erythromycin, we have been able to show that physical integration occurs at a site near the unreplaced recipient allele and that segregation is the probable origin of all substituted haploids. Other interesting properties of this marker, undoubtedly reflecting its unusual physical nature, were encountered in the course of this study, the results of which we shall now set forth.

MATERIAL A N D METHODS

Strains and genetic markers: The principal recipient strain employed was the Challis strain of Streptococcus sanguis. ‘The original donor strain was obtained from DR. ROMAN PAKULA in 1962, and contained, as a consequence of selecting three successive spontaneous mutations, markers for erythromycin (ery-ra), streptomycin ( s tn-43) and nwolbiocin (nov-7-20) resistance. The ery-r8 marker was transferred to the SIII-1 strain of Streptococcus pneumoniae (pneumococcus) and subsequently from this interspecific transformant into the R6 strain of S. pneumoniae. In both steps a stable transformant was selected having the high level of erythromycin resistance characteristic of eryr8 The R6 ery-r8 strain served as a donor in the transformation of two pneumococcal strains isogenic except for the hez marker: R6 (he&) and R6x (hex-).

Media and growth: Challis cells were grown in Medium 1 (ROTHEIM and RAVIN 1961), except that Pk containing NaCl at a final concentration of 0.65% (RAVIN and DESA 1964; METZER and RAVIN 1972) Wac used for preparing competent cells and transforming them. The medium for growth and transformation of pneumococci was NS (ROTHEIM and RAVIN 1961). The plating medium for all strains was Medium 1 + 1.5% agar. Growth was monitored either by plating diluted samples to determine numbers of cdony-forming units (c.f.u) or by measuring optical density in a Coleman Model 7 photo-nephelometer.

TRANSFORMATION-INDUCED MERODIPLOIDS 423 Transformation: The methods used for preparing transforming DNA, for obtaining competent

Challis cells, and for transforming them have been previously described (CHEN and RAVIN 1966; RAVIN and DESA 1964; ROTHEIM and RAVIN 1961). The method used for preparing competent cells of pneumococcus and for transforming them was described by DEJIDISH and RAVIN (1974). Selection of transformants acquiring the ery-r8 marker will be described under RESULTS.

Plating in agar mta in ing 100 pg/ml streptomycin and 10 p g / d novobiocin selects for str-r43 and nov-rlU transformants, respectively.

Selection was carried out by either the direct or overlay method. In the former, a population previously exposed to transforming DNA was allowed to grow and express its newly acquired phenotypes before being plated in selective media. In the latter method, the population was plated immediately in non-selective media and, after phenotypic expression had occurred, an agar overlay was added to select transformants.

Ultraviolet irradiation of DNA: 1 ml containing 23 pg of the transforming preparation was added to the bottom of a 100-mm Petri dish. The contents of the dish were exposed for various lengths of time to irridation from a Hanovia germicidal lamp, the energy output of which at 254 nm was measured by means of an IL-254 Germicidal Photometer (International Light, Inc.). A sample of the irradiated DNA solution was diluted into the transformation reaction mixture to obtain the desired final concentration.

Treatment of cells: Thymidine deprivation was achieved by using a thy- strain of Challis requiring 50 gg/ml of thymidine for growth at a normal rate. The thy- marker had been introduced previously into Challis from the Wicky strain of Streptococcus sanguis in which it originated, and the ery-r8/+ merodiploid genome was produced in this thymidine-requiring strain when effects of thymidineless death on segregation were to be determined. Rifampin (or RIF) was added at a final concentration of 5 pg/ml. When used on competent cells, it was added 5 minutes before DNA which was allowed to act for 20 minutes before addition of pancreatic DNase I and incubation for five more minutes; a t this time the cells were centrifuged and washed before allowing further growth and phenotypic expression of newly acquired DNA. When RIF was used otheiwise, the total length of treatment was 30 minutes. Acridine orange (or AO) was used at a concentration of 5 pg/ml in liquid media and 50 pg/ml in agar media. In liquid media containing an A 0 concentration of 5 pg/ml, the growth rate was depressed, the yield in number of CFU being 50-80% that of an untreated control which had undergone approx- imately 11 generations. RIF and AQ were obtained from CalBiochem.

Segregation: To study the frequency of segregation from the ery-r8/+ strain, a culture was highly diluted so that a 0.01-ml sample contained only a few cells. Each of ten such samples was inoculated into a tube containing 1 ml of Med 1. These cultures were grown at 37" to obtain the desired cell densities. The cultures were then plated in agar containing 0.05 and 0.25 pg/ml erythromycin; colony counts from the former gave the total number of ery-rd/+ cells, while counts from the latter gave the number of ery-r8 segregants. When err+ (erythromycin-sensitive or wild-type) segregants were t o be obtained, a plating was also carried out in medium devoid of antibiotic. Colonies were picked from these plates, and tested by velvet replication onto a series of agar plates varying in erythromycin concentration. Control colonies of wild-type err+, ery-r8/+ and ery-r8 were tested at the same time: after 24 hours' incubation at 37", e r y f replicas were negative, ery r8/+ and ery-r8 replicas positive on agar containing 0.05 pg/ml erythromycin; ery + and ery-r8/+ replicas were negative, ery-8 replicas positive on erythromycin concentrations between 0.25 and 100 pg/ml.

RESULTS

Properties conferred by the ery-r8 marker: The original strain carrying the ery-r8 marker has an extremely high level of resistance to erythromycin, plating with 100% efficiency in agar containing up to IO0 pg/ml of the antibiotic. The cry-r-8 strain grows at the same rate as the wild-type strain in Medium 1 or Pk free of antibiotic (generation time = 30-35 min) , and can be rendered competent

424 A. W. RAVIN A N D M. MA

to undergo transformation with the same high efficiency as the wild-type strain (see Table 8).

The original strain has been perfectly stable, never reverting to a lower level of resistance in the hundreds of transfers it has undergone in 13 years. The strain has also been quite stable during growth in the presence of 50 pg/ml AO, which hase been shown to cure coliform bacteria and streptococci of autonomously replicating episomes (HIROTA 1960; WATANABE and FUKASAWA 1961 ; CLEWELL et a2. 1974). Eficiency of transformation by DNA bearing the ery-r8 marker:

1. Rate of phenotypic expression: Wild-type (ery + str 4- nov +) cells were exposed for 20 min to 1 pg/ml DNA from the triply marked donor strain (ery-r8 str-r43 nov-rl0) and the culture diluted ten times in fresh medium after DNase treatment. Dilution was necessary in order to extend the length of the exponen- tial phase of growth. The culture was plated at hourly intervals in non-selective media to obtain the total number of c.f.u. At the same times platings were carried out in media containing 100 pg/ml streptomycin to select for str-r43 transformants and in media containing four different concentrations of erythromycin: 0.05, 0.25, 10 and 100 pg/ml. The lowest erythromycin concentration was only five times above that which will support 100% plating efficiency in the wild-type recipient strain. I t is selective for transformants, however, so long as the competence of the populatioc is high (20-100% in our experiments) and the plating medium carefully adjusted to pH 7.3.

The rate of appearance of streptomycin- and erythromycin-resistant transformants is shown in Figure 1. Phenotypic expression of streptomycin resistance was completed in one hour since the rate of growth of str-r transformants was identical to that of total c.f.u. after that time. When 0.05 pg/ml erythromycin was used to select ery-r transformants, their expression was completed in one hour, at which time they were nearly twice as frequent as str-r transformants. Resistance to 10 pg/ml erythromycin was not yet complete in 4 hours since at that time the n u m b s of transformants selectable on this concentration was still rising faster than the total population. In subsequent experiments (not shown), we found that the frequency of transformants selectable on 10 pg/ml reached a maximum in 5 to 8 hours, at which time their number was equal to that obtained on 0.25 pg/ml. No transformants capable of growing on 100 pg/ml were detectable within 8 hours. Transformed cultures that were transferred by dilution into fresh media eventually contained such highly resistant cells. however.

Transformed colonies selected at each of the erythromycin concentrations were picked and tested by velvet replication onto a series of agar plates containing either 0.05, 0.25, 10 or 100 pug/ml erythromycin. Of 98 colonies picked from 0.25 pg/ml and 49 from 10 pg/ml, all were capable of replicating to the highest concentration of erythromycin. Of 49 colonies isolated from 0.05 pg/ml, none replicated on erythromycin concentrations of 0.25 pg/ml or above, although a fcw colonies did contain a small number of cells that formed slowly growing papillae within the replicated area on these high concentrations of erythromycin. More will be said about these papillae below. We concluded that there were two

TRANSFORMATION-INDUCED MERODIPLOIDS 425

106

- io5 E \ cn I- z I a a

a a

$ 104 z

I-

L L 0

E 103

4 m

z

10'

10'

P [c.f. u.1

P (32')

k ' , , , ,

108

0 1 2 3 4 5 TIME (HRS) ALLOWED FOR EXPRESSION

FIGURE 1 .-Kinetics of expression of donor markers str-r43 and ery-rg after 20-min treatment of ery + str + recipients by 1 pg/ml donor DNA. Number of colony-farming units (c.f.u.) determine1 by plating on non-selective agar (P) at times indicated. Number of erythromycin- resistant transformants determined by plating in agar containing 0.05 (E,,o,), 0.25 (E,,,25) and 10 (Elo) gg/ml erythromycin. Number of streptomycin-resistant transformants determined by plating in agar containing 100 pg/ml streptomycin (S). Number indicated in parentheses = generation time in minutes determined from ultimate slopes.

classes of transformants produced by the donor ery-r8 marker, one marked by very low resistance and one by a resistance identical to that of the donor strain. Expression of the highest level of resistance conferred by the ery-r8 marker was relatively slow, however. For this reason, ery-r8 transformants like the donor were thereafter selected by challenging with 0.25 pg/ml erythromycin after allowing 2 to 3 hours for phenotypic expression.

2. Dependence of transformant frequency upon DNA concentration: Samples of a competent wild-type recipient culture were exposed for 20 min to various concentrations of donor DNA, and then plated after 3 hours of growth in streptomycin (100 pg/ml) agar and in agar of two different erythromycin concentrations, 0.05 and 0.25 pg/ml. The results are shown in Figure 2. The number of str-r transformants was directly proportional to DNA concentrations up to 0.05 pg/ml (slope or k = 1.1 in a log-log plot). The number of ery-r transformants


I P

10' -~--ii-T------mT7-y

0.1 1 1 '

0.0 1 DNA CONCENTRATION ( p g / m l )

FIGURE 2.-Dependence of transformant nuniber on concentration of donor DNA ery-r8 str-r43. ery-r8/ f merodiploids, ery-r8 and str-r43 stable transformants determined, respectively, by plating in 0.05, 0.25 p g / d erythromycin and 100 ag/ml streptomycin. K = slope of line at point indicated (expressed as log no. transformants/log DNA concentration), Circles = e r y - r d / f ; squares = str-r43; triangles = ery-rg. Closed and open forms of same symbol represent data from duplicate experiments.

selected in 0.05 pg/m1 erythromycin was similarly proportional to DNA concentration, but was from 1.5 to 2 times more €requent than str-r transformants at all DNA concentrations. The number of ery-r transformants selected on 0.25 pg/ml was extremely low and exhibited a peculiar relation to DNA concentration. At very low DNA concentrations ( <0.05 pg/ml) , transformant number was proportional to DNA concentration and about 500 times lower than the number of str-r transformants. At higher DNA concentrations, however, transformant number rose as the square of the increase in DNA concentration (k = 2.0), until the saturating level of DNA was reached. At the highest DNA concentration employed (1 pg/ml) , the number was about 70 times less than that of str-r transformants. Clearly, the frequency of the two types of transformants distinguished in the preceding section responded differently to donor DNA concentration.

TRANSFORMATION-INDUCED MERODIPLOIDS 42 7

While low-level and high-level resistance transformants could be produced by single molecules of transforming DNA, the probability of transformation to the highly resistant type was significantly enhanced by the cooperative action of two molecules of DNA bearing the ery -d marker. The peculiar dependence on DNA concentration of the production of ery-r8 transformants like the donor was confirmed in several separate experiments.

Merodiploid nature of transformants produced at high efficiency: Transform- ants produced by the ery-r8 marker with high efficiency exhibited a resistance to erythromycin a couple of orders of magnitude less than that of the donor strain. There were several indications, moreover, of their instability. Not only were numerous papillae observed, as noted above, when transformed colonies selected at low erythromycin concentrations were picked and tested by velvet replication onto high erythromycin concentrations, but cultures of isolated colonies grown in liquid media were found soon to contain mixtures of three types of cells: unstable, poorly resistant parental-type cells; stable erythromycin-semitive cells like the original wild-type recipient; and stable erythromycin-resistant cells like the original ery-r8 donor (Table 1 ) . In such mixtures the proportions of sensitive and highly resistant cells varied in different cultures, but the ery-r8 was always found along with the ery+ type. We concluded that the poorly resistant transformant produced with high efficiency was in fact a diploid (ery-r8/+) in which the homologous recipient allele (+) coexisted with the donor allele ( r8 ) and, because of the strong dominance of the sensitive allele, the transformant was only weakly resistant to erythromycin. The ery-r/+ strain was only partially diploid (merodiploid) , however, since it could be subsequently transformed by the recessive str-r43 marker with the same “one-hit” kinetics as a wild-type recipient.

That the highly resistant segregants were identical to the original ery-r8 donor was shown not only by their similar level of resistance to erythromycin, but also by the fact that DNA prepared from them exhibited transforming activities similar to that of DNA from the original donor and to those of DNA preparations extracted from highly resistant transformants produced at low efficiency. Table 2 (Experiment 11) compares the activities of DNA preparations from three

TABLE 3

Detection of segregants in cultures of ery-r8/+ transformants

Total no. cells/ml resisting Wg/ml ery % ery-r8

Culture no.

Colonies isolated from antibiotic-free medium

No. No. No. % e r r + tested ery f ery-r8 (2)/(1)

(1) ( 2 ) x 100

2.7 x 1018 2.9 x lW 5.0 x 106 2.8 x 10s 3.6 x 108 2.0 x IO6 5.9 x 108 5.3 x 108 1.9 x 107 4.3 x 10s 4.2 x 108 6.1 x IO5 4.5 x I@ 2.8 x 108 8.1 x 106 2.1 x I@ 1.1 x l@ 4.7 x 10’6

1.7 0.6 3.6 0.1 2.9 4.3

335 6 5 1.7 10 1 0 10.0

9 3 2 . 1 - 0.4 232 1 - 0.4 525 16 8 3.0 581 2 17 0.3

“-” means not checked. Culture no. 6 had been grown in presence o,f 5 pg/ml AO.

428 A. W. RAVIN AND M. M A

TABLE 2

Transforming efficiencies of DNA prepmotions from strains eryr8 str-1-43 and ery-r8/+ str-1-43

Expt. __ I

I1

I11

IV

V

- ~

DN 4 prep

ery-i-8 str-r43 ery-r8/+ tr.1 str-r43

ery-r8 str-r43 ery-r8 tr.1 ery-rg tr.2 e r y d seg. 1 ery-r8 seg. 2 e r y d seg. 3 ery-r8/+ tr.1 ery + seg. 1 ery-r8 str-r43

ery-r8 tr.3 str-r43 ery-r8 tr.4 str-r43 e r y d tr.5 str-r43 ery-r8/+ tr.1 strv-43

ery-r8 str-r43

ery-r8 tr.3 str-r43

ery-r8 tr.6 str-r43

ery-r8/+ tr.2 str-r43

ery-r8 str-r43 ery-rlb str-r43 ery-r17 str-r43

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.4 0.04 0.4 O.w 0.4 0.0’4 0.4 0.M 1 1 1

No. transformants/ml

srl--r43 wy-rS-S/+ ery-rS ~ _ _

2.5 x I@ 5.7 x IO4 1.3 X 10’ 1.7 x 104 0.9 x IO4 1.5 X IO1 1.6 x 105 2.9 x iolj 1.2 x 103

- 1.2 x 1015 0.6 x 10.3 - 0.6 x 105 0.1 x 103 - 1.7 x 105 0.9 x 103 - 1.5 x 105 0.5 x 103 - 1.1 x 104 0.6 x loll - < I <I

6.4 x 105 9.1 x 105 1.9 x 103 5.4 x IW 7.8 x IO“ 2.4 x 103

3.3 x 10s 6.1 x 105 1.9 x 103

4.1 x 105 5.5. x 105 9.4 x IO?

4.4 x 1015 1.3 x 106 4.1 x 103

3.3 x 10s 1.8 x 105 2.4 x 1012

4.4 x 105 9.3 x 105 6.4 x 103 4.4 x 105 7.5 x io; 3.0 x 103

- 1.7 x 105 0.5 X lo3

1.6 x IOG 3.3 X lo6 2.1 X 1w 1.6 x I@ 2.6 X 10l6 0.9 X IOt4

1.2 x 10’6 6.2 x IO5 9.3 X IO8*

2.6 x 1Ori 4.9 X lo4 3.6 X 10’

3.0 x 10/1 7.4 x IO* 4.1 X IO1

4.8 x IO4 1.1 x IO5 6.6 X IO1

2.8 x 104 1.8 x 104 4.4 X loo

2.7 x 1015 7.6 x IO6 3.3 X IO3

2.3 0.5 1.8 - - - - - - - 2.1 1.6 1.4 1.4 0.5 1.9 1.9 1.3 2.5 2.9 2.3 0.5 0.6 2.1 2.3 2.8

err-rS

ery-rS/+

0.008 0.002 0.004 0.003 0.005 0.002 0.005 0.003 0.001

0.W6 0.m3 0.002 0.003 0.002 0.003 0.0007 0.002 0.0006 0.m3 0.00106 0.001 0.0002 0.007 0.004 0.004

-

Experiments I-V were done with different competent batches of ery + str -k cells. tr.1-tr.5 and seg. l-seg. 3 mfer to independent transformants or segregants, respectively, of genotype indicated. ery-r8 tr.6 was transformant obtained with limiting conc. (0.02 pg/ml) DNA; ery-rS tr.1- tr.5 were obtained with a saturating conc. (1 pg/ml) DNA. ery-ri‘b and ery-r17 are independent, spontaneous erythromycin-resistant mutants; in their case, number ob transfomants/ml. under columns headed “ery-r8/+” and “ery-rb” refer, respectively, to transformants selected on 0.05 and 0.25 pg/ml. erythromycin.

independent ery-r8 segregants with those of two independent transformants and that of the original donor. Each DNA produces poorly resistant transformants with high efficiency and highly resistant transformants with lower efficiency. In addition, the DNA obtained from an ery+ segregant was shown (Table 2, Experiment 11) to be without activity in inducing poorly or highly resistant ery-r transformants. In this respect the DNA of the ery + segregant was identical to that of the original sensitive recipient.

Although merodiploid cultures upon continued growth contain ery + and ery-7-8 segregants, the proportions of segregants can be kept low by plating at a low concentration of erythromycin (0.05 pg/ml) and isolating an ery r8/+ colony to start a new culture. In this way large populations can be grown for the


purpose of extracting DNA and the proportion of segregants (checked by plating prior to cell lysis) kept below 3%. DNA preparations obtained from ery-r8/+ cultures in this way were tested on the wild-type recipient strain, and the results are shown in Table 2 (Experiments I, 11,111, and IV) . Some of the merodiploid strains contained the str-7-43 marker, which had been transferred into them prior to extraction of DNA; in this way, a reference marker was available for evalu- ation of transforming activity. It may be seen that the production of poorly resistant transformants by the ery-r8 marker was consistently more efficient than that obtained with DNA from the ery-r8+ merodiploid. Hence, the presence of the + allele lowered the transforming activity of the r8 marker. When a reference str-r marker was present in the DNA preparations, it was possible to show that the difference in efficiency between the ery-r8/+ and ery-r8 DNA preparations was of the order of four to five times and independent of DNA concentration (Table 2, Experiment IV) . This evidence strongly indicates that both the r8 and 4- alleles can be transmitted by the same molecule of DNA and are, therefore, linked in the ery-r8/+ genome.

Stability of the ery-r8/+ merodiploid: The rate of growth of merodiploid cultures in the absence of erythromycin was indistinguishable from that of either ery 4- or ery-r8 cultures. With prolonged growth, however, the proportion of segregants in the culture appeared to increase. Accumulation would indeed be expected if there were a constant probability of segregation per bacterial generation and fixation at the ery-r8 and ery + states. To obtain a more accurate esti- mate of the frequency of segregation (in contradistinction to segregants) , a modified statistical fluctuation test was performed, as described under MATERIALS

AND METHODS. Ten tubes, each receiving a small number of ery-r8/+ cells from a culture having low incidence of existing segregants, were plated after many generations had elapsed onto media containing 0.25 p/ml erythromycin, selective for ery-r8 segregants, and 0.05 bg/ml erythromycin, permissive for the growth of merodiploids. The results are shown in Table 3. The incidence of ery-r8 cells was fairly uniform from tube to tube and several orders of magnitude higher than that of spontaneously arising erythromycin-resistant mutants (see below) , indi- cating that segregation rather than mutation was the cause of their occurrence. The incidence was also significantly higher in cultures that had undergone a longer period of growth. From these results we calculated the actual frequency of segregation as 2 x segregations per bacterium per generation, using the equation: s(segregation rate) = 2(loga2) (S,/N, - S,/N,)/g, where SI and Sz,

TABLE 3

Frequency of segregation from ery-r8/+ to ery-r8

Percent Avg. Avg. ers-rS no. cells final no. Percent ery-rX cells present

in inoculated cells Tube no. Expt. parental per tube per tube No. culture (1 ml) ( 1 ml) 1 2 3 4 5 6 7 8 9 1 0 A v g .

1 1.9 6.2 1.3 x 108 .I1 .08 .@3 .08 ,139 .(E9 .(E3 .IO .08 . I1 .Of3 2 1.7 5.8 7.0 x 106 .01 .02 .013 .04 .03 .I33 .@+ .03 .03 .I% .03

43 0 A. W. RAVIN AND M. M A

and N I and N , are the number of segregants and total bacteria at times 1 and 2 respectively, and g is the number of generations between times 1 and 2.

The frequency of segregants was not strongly affected, however, by treatment 01 merodiploid cultures with either acridine orange or thymidine deprivation, both of which are known to be effective eliminators of episomes (HIROTA 1960; WATANABE and FUKASAWA 1961; CLOWES, MOODY and PRITCHARD 1965). The effect of thymidine deprivation was examined in thy - ery-r8/+ cultures containing initially less than 1 % segregants. Despite over tenfold declines in c.f.u. after several hours in the absence of thymidine, no significant increase was observed in the proportion of either ery-r8 or ery+ segregants. The effect of acridine orange on segregation was equally negative. Liquid cultures grown for 20 generations in 5 pg/ml A 0 showed no significant increase in the proportion of total segregants, nor any predominance of ery + segregants (culture no. 6, Table 1 ) . Moreover, when an ery-r8/+ culture was plated in an agar medium containing 50 pg/ml AO, the proportion of colonies found to be ery + (1/347) was not higher than that found in a control plating devoid of A 0 (2/397). An AO-induced increase in ery+ segregants would have been expected if the ery-r8/+ merodiploid contained the r8 allele in an episome unintegrated into the chromosome carrying the + allele (CLEWELL et al. 1974). These results would be consistent, on the other hand, with the interpretation that the r8 and f alleles are linked in the merodiploid genome.

Production of stable transformants: As already pointed out, the highly resistant cells produced by transformation of sensitive recipients with donor DNA carrying the ery-r8 marker were identical phenotypically and genotypically to ery-r8 segregants and to the original donor strain (Table 2, Experiments I1 and 111). Moreover, like the original donor and the ery-r8 segregants, the ery-r-8 transformants were highly stable during periodic transfer.

On the other hand, stable ery-r8 transformants appeared to be produced by a different mechanism than that giving rise to ery-rS/+ merodiploids. Not only was the probability of an ery-r8-bearing molecule producing a merodiploid over two orders of magnitude higher than the probability of its producing a stable transformant, but there was evidence of cooperativity between multiply infecting molecules in the production of stable transformants and none in the case of merodiploids (Figure 2). That the cooperative effect required specifically ery-r8

TABLE 4

Specificity in the cooperatiuity of en-1-8 markers in the production of stable transformants

Donor DNA No. transformants/ml ery-rS

Markers (pg/ml) str-r43 ery-rS/+ ery-rS ery-rX/ f

str-r43 ery-r8 1.0 6.9 X 1015 1.3 x 10’6 8.7 x 103 6.6 x lot3 str-r43 ery-r8 0.02 1.5 X 10. 4.0 x 104 1.0 x lo1 e.; x l o r 4

“.“2] 2.3 X lo5 3.2 x 104 2.0 X IO1 6.2 x 10-4 F - r 4 3 r - r 8

[str-r43 ery-s 1.0 J str-1-43 ery-s 1 .O 3.1 x 105 < io3 <lo1 -


molecules may be seen in the results of the experiment summarized in Table 4. In this experiment the effect of adding ery i- markers to cells receiving single molecules of ery-r8 DNA was compared with the effect of multiple infection by ery-r8 markers. The results showed that on ly multiple infection by DNA specifically containing the ery-r8 marker enhanced the production of stable ery-r8 transformants.

To determine whether stable ery-r8 transformants produced by multiple infection were different from those produced by single infection, transformants obtained with 0.02 and 1 pg/ml DNA were isolated and compared with respect to their level of erythromycin resistance and the transforming activity of DNA extracted from them. As for level of resistance. no difference could be detected when such strains were plated an increasing concentrations of erythromycin; both types of transformants exhibited 100% plating efficiency at concentrations up to 100 pg/ml but not 200 pg/ml. Examining the genetic activity of DNA extracted from them, no difference could be detected in respect to efficiency of inducing ery-rd/+ merodiploids and stable ery-r8 transformants in ery + recipients (Table 2, Experiment IV) .

Sensitivity of the production of stable ery-rg transformants t o UV-irradiation of DNA: One hypothesis to account for the large difference in frequency between stable ery-rg transformants and ery-r8/+ merodiploids would be that the latter are produced by the same kind of process by which HE markers in general are integrated, whereas the formed occur by the distinctly different process by which LE markers are integrated. Since LE markers are known to be considerably more sensitive to ultraviolet irradiation than HE markers ( EPHRUSSI-TAYLOR. SICARD and KAMEN 1965; LACKS 1965), it was of interest to compare the irradiation sensitivities of merodiploid and stable transformant production. In Figure 3 are showfi the results obtained by applying various doses of ultraviolet light to the triply marked nov-rZ0 str-r43 ery-r8 DNA and using the irradiated DNA at a concentration of 1 pg/ml to transform wild-type recipients in the usual manner. It should be pointed out that in these experiments the rate of phenotypic expression of str-r43, ery-r8/+ and e r y d transformants was found to be unaffected by irradiation of the DNA.

Reference to Figure 3 reveals a correlation between the intrinsic efficiency of transformation of a marker and its sensitivity to U.V. The nov-rZ0 marker was the most efficient and exhibited the lowest sensitivity. Production of ery-r8/+ merodiploids was about as efficient as the production of nou-rZ0 transformants, and its sensitivity to UV-irradiation was about the same. Production of str-1-43 transformants showed consistently half of the efficiency of nov-rZ0 transformations, and its UV sensitivity was distinctly greater. Production of stable ery-r8 transformants was the most sensitive to UV. However, its sensitivity was not as great as one might have anticipated on the basis of its efficiency. A possible explanation was that at 1 pg/ml DNA considerable cooperativity existed in the production of stable transformants (see Figure 2), and that UV damage to DNA molecules was interfering with this cooperativity and not lowering the efficiency of a single molecule producing a stable transformant relative to its producing a

432 A. W. RAVIN AND M. MA

marker efficiencies \. \A-

nov-r lo =LO cry-"++ =OB s t r - r 4 3 =04 ery- r 8 =0.006

0 3 6 9 12 MINUTES OF UV I R R A D I A T I O N

FIGURE 3.-Inactivation of donor markers by ultraviolet irradiation of DNA ery-r5 nou-rlU sir-r43. Recipient cells exposed to 1 fig/ml DNA. c.f.u. = colony-forming units. Intensity of irradiation = 6 . e x IO15 ergs/cmz/min. Marker efficiencies determined relative to nov-rl0 in unirradiated DNA.

nierodiploid. To test this possibility, cells were transformed by either 1 or 0.02 pg/ml of DNA, which had either been unirradiated or irradiated with a single dose of UV. The results are given in Table 5. Cooperativity in the production of stable ery-r8 transformants was evident by the higher transforming efficiency at the saturating concentration (1 pg/ml) than at the limiting concentration (0.02 pgJml) of DNA. No significant difference in transforming efficiency was seen between the two DNA concentrations for the production of either ery-r8/+ merodiploids or stable nou-rl0 and str-r43 transformants. The production of merodiploids and stable nou-rl0 transformants showed similar transforming efficiencies and similar sensitivities to the UV dose employed, regardless of the concentration of DNA with which the cells were treated. The production of stable sZr-r43 transformants was half as efficient as that of merodiploids and nov-rlO


TABLE 5

Sensitiuity to ultrcruiotet irradiation of two types of transformnts produced by ery-r8 marker

Transformant Initial Percent DNA Genetic type transforming surviving

conc.* marker produced efficiency+ activity$

1.0 noNu-rl0 str-r43 ery-r8 ery-r8

str-r43 ery-r8 ery-r8

0.02 rrou-rl0

stable stable merodiploid (ery-r8/+) stable stable stable merodiploid (ery-r8/+) stable

1 .o 0.4 0.9 0.0017 1 .o 0.5 0.9 0.0006

30.6 12.0 27.5 9.6

34.7 15.8 34.1 35.8

*Concentration of DNA to which cells were exposed. +Initial transfanning efficiency is basd upon activity d unirradiated DNA; efficiency of

$ Dose of UV = 6 min. exposure to 10,813 erg/cmZ/sec. nou-ri0 marker is set at 1.0 and efficiencies ob other markers are normalized to this value.

transformants. It was also two to three times more sensitive to the UV dose employed. While at the higher concentration of DNA the production o€ stable ery-r8 transformants was the most sensitive to U V irradiation, just as seen in Figure 3, transformation to the stable ery-rd state was no more sensitive to irradiation than transformation to the merodiploid ery-r8/+ state when a limiting concentration of DNA was used. It follows, therefore, that ultraviolet light can damage the ability of ery-r8-bearing molecules to cooperate in effecting a stable transformation, but that the intrinsic efficiency with which a sil.-gle one of these molecules can effect a stable transformation is not more sensitive to UV than the efficiency with which it can produce a merodiploid. A corollary of this conclusion is that the production of stable transformants does not occur by the process of integration peculiar to LE markers.

Transformation by the ery-r8 marker in a hex- recipient: In S. pneumoniae, hex- mutant strains exist which fail to discriminate against low efficiency (LE) markers in homospecific DNA. As a further test to determine whether the ery-r8 marker were integrated by two distinct processes, one highly efficient (HE) leading to merodiploids and the other poorly efficient (LE) leading to stable transformants, a hex- recipient was transformed by DNA carrying the ery-r8 marker. Were stable ery-r8 transformants produced in hex+ strains by an LE process, they would occur with high efficiency in a hex- strain. In order to remove heterologous DNA, the ery-r8 marker was first transferred from S. sanguis into S. pneumoniae as described in MATERIALS AND METHODS.

DNA extracted from pneumococcal R6 ery-r8 cells was presented to hex+ and hex- mutant cells of strain R6. In the same experiment an R6 DNA preparation containing an HE (str-r4l) and an LE (rif-rl2) marker was tested on similar cells. Transformants were selected by the direct method after 2 hours of phenotypic expression, using 100 pg/ml streptomycin, 1 pg/ml rifampicin, 0.075 p g / d erythromycin and 0.25 pg/ml erythromycin folr str-r#l, rif-rl2,


TABLE 6

Trnnsformation by the ery-r8 marker in hex+ and hex- recipients

Relative efficiency of markers in DNA prep. no.

Recipient

1 rif-rIZ

2 ery-r8

str-r4i ery-r8/+

R6 hex+ R6 hex-

0.13 0.87

0.003 O.oD5

DNA preparation no. 1 = R6 str-r41 r i f d 2 ; no. 2 = R6 ery-r8. These two preparations showed similar activities for str-r4i and ery-rJ/+ transformations at

concentration employed (1 pg/ml).

ery-r8/+ and ery-r8 transformants, respectively. It should be noted that a slightly higher concentration of erythromycin is necessary to select for merodiploids in pneumococcal strains because of the slightly higher level of resistance to erythromycin manifested by this species. From the results recorded in Table 6, it may be seen that the hex- strain shows hardly any discrimination against the rif-r12 marker, which behaves as a typical LE marker in transforming the hex+ strain. Nevertheless, the production of stable ery-r-8 transformants occurs with the same low efficiency relative to ery-r8/+ merodiploids in hex+ and hex- recipients. This result cannot be due to tight linkage between hex+ and ery-r8 in the donor DNA, since hex- recipients transformed by the ery-r8 marker were subsequently found to retain their lack of discrimination against pneumococcal LE markers rif-rl2, opt-r2 and stg-rF. It follows that the normal hex function is not the cause of the low frequency of stable ery-r8 transformants.

Specific enhancement of stable ery-r8 transformations by rifampin: One possibility to account €or all of the results is that the only kind of transformation produced by a molecule bearing the ery-i-8 marker is a merodiploid. This transformation occurs with the high efficiency typical of high-efficiency (HE) markers like nor?-rlU. In this hypothesis, all ery-r8 cells arise by segregation of the wild- type allele from initially merodiploid cells. In support of this explanation is the relative frequency of stable ery-rg cells observed three hours after treatment of wild-type recipients with a limiting concentration of ery-1-8-bearing DNA. This frequency has ranged from 1.3 x to 3 x per cry-r8/+ merodiploid in the culture (Figure 2 and Table 4), which is a range of values encompassing the frequency of ery-r8 segregants (2 x 1 O*/merodiploid/generation x c.4 generations) expected on the basis of the spontaneous segregation rate previously determined.

If this hypothesis were correct, an agent that stimulated the production of stable ery-r8 transformants would do so by stimulating segregation from ery-r8/+ merodiploids. RIF is an agent that specifically stimulates transformation by heterospecific DNA and certain homospecific markers (DEDDISH and RAVIN 1974). Its effect on the production of stable ery-r8 transformants is shown in Table 7, which presents the results of treating recipient cells with RIF while being transformed by donor DNA carrying the nou-rl0, str-r43 and ery-r8

TRANSFORMATION-INDUCED MERODIPLOIDS

TABLE 7

Effect of rifampin on the frequency of LE ery-r8 transformants Method I. Direct selection: str+ err+ cells x str-r43 ery-r-8 DNA

435

str-r43 ery-r8/+ ery-rS

+ RIF + RIF + RIF RIF Total cells - -_ -

Expt. treatment X 1 0 7 / ~ l /lo3 cells -RIB /I@ cells - RIF / I F cells --IF

1 - 3.9 5.9 5.4 7.4

2 - 0.86 6.2 5.3 6.8

3 - 2.2 5.9 8.6 5.4

+ 0.51 5.7 1.0 3.5 0.7 19.6 2.6

+ 0.25 4.6 0.7 8.9 1.7 18.5 2.7

+ 0.29 7.2 1.2 16.2 1.9 28.7 5.3

Method 11. Overlay method: nou+ strf err+ cells x nou-7-10 str-r43 eryr8 DNA nou-rl0 str-r43 ery-r8 - + RIF 4- RIF + RIF

RIF Total cells - - - Expt. treatment X IoB/ml perml --IF perml -RIF perml --IF

1 - 2.4 1.7 x 1W 7.2 X 10" 2.0 x 1013

2a - 3.2 ND 9.2 x 104 2.4 x 103

2b - 2.6 ND 1.9 x Io" 7.5 x loo

+ 1.4 1.7 x 105 1.0 11.6 x 104 1.6 12.6x 103 6.3

+ 1.7 ND 1.5 x lo5 1.6 1.4 x 104 5.8

+ 2.1 ND 3.7 x 103 1.9 4.0 x 101 5.3

"ND" means "not done". 1 pg/ml DNA used in all exwriments except 2b in which 0.@2 p g / d was used.

markers. Using the direct method of selection, one could detect the effect of RIF in the diminished size of the transformed population at the time of plating. How- ever, RIP had little or no effect on the frequency of nov-rl0, str-r43 or ery-r8/+ transformants, but caused a large enhancement of ery-7-8 transformants. The overlay method of selecting transformants was used to obviate possible specific effects of RIF on the growth rates of the different types of transformants. Unfortunately, ery-r8/+ cannot be selected by this method because of interfer- ence by untransformed cells, which are allowed to grow in the agar media during the period allotted for phenotypic expression. Nevertheless, use of this method of selection showed that the stimulating effect of RIF on the frequency of ery-r-8 transformants cannot be accounted for by some differential effect on growth. It was also noted that RIF pr'oduced its enhancing effect on the production of ery-r8 transformants not only at a DNA concentration that causes a cooperative effect ( 1 pg/ml) but also at one that does not (0.02 pg/ml) . Hence, RIF does not act to increase the cooperativity of ery-r8 molecules in inducing stable transformants.

To check whether RIF acts by increasing the probability of segregation of ery-rg cells from ery-r8/+ merodiploids, we performed the following test. A merodiploid culture containing about 2% segregants was diluted so that 0.03 ml vr7ould contain, on the average, 4 c.f.u. Inocula of this size were made into 10 tubes containing 1 ml of medium. These cultures were then incubated for 9 hrs at 37",


O.I8 0. I6

ar w a 0.08

0.06

0.04

0

2 3 4 5 6 7 8910 15 NO. OF C . E U X IO’ I N CULTURE

FIGURE 4.-Per cent ery-r8 segregants in cultures treated (squares) or not treated (circles) with 5 pg/ml rifampin for 30 min. Procedure of experiment described in text.

at which time four of the cultures received 5 pg/ml RIF. After a half hour of iurther incubation all of the cultures were centrifuged, washed and resuspended in 1 ml fresh medium. The cultures were incubated to permit two to three generations of growth (for phenotypic expression of newly arising segregants), and then plated to determine the number of merodiploids and stable, highly resistant segregants. The results are shown in Figure 4. which indicates the percentage of stable segregants as a function of the number of c.f.u. in the culture. Not all cultures contained the same number of c.I.u., probably because of the great vari- ability in the onset of growth in cultures started from small inocula. However, it was clear that populations treated with RIF showed a 2- to 3-fold higher frequency of segregants than did untreated populations of similar size. It was also clear that the proportion of segregants in the untreated cultures increased with growth at a rate similar to that calculated above. In repeats of this type of experiment, RIF was always found to have a stimulatory effect on segregation although the extent of stimulation varied from one and a half to three times.

Ery-r8 is linked to a ribosomal ery-r marker: The unusual behavior of the ery-8 marker raised the possibility of its belonging to a locus different than the ery locus in which mutations causing ribosomal resistance to erythromycin are known to occur (RAvIN, ROTHEIM and COULTER 1969). To check on this possibility, the ery-r2 marker, which is a typical HE marker and has been shown to determine ribosomal resistance, was transferred uia interspecific transformation from its strain of origin (Strep. pneumoniae, SIII-1 strain) into the Challis strain. This Challis ery-r2 strain was made competent in the usual manner, and then transformed with Challis DNA containing the str-r43 and ery-r8 markers. The


transformed culture was plated in non-selective media immediately after addition of DNase, and the plates received a second agar layer 2 hours later. The second layer was either selective for streptomycin-resistant transformants or non- selective. From the numbers of colonies appearing on these plates, the efficiency of str-r transformation was calculated to be 35 per 100 c.f.u. One hundred and sixty-seven colonies were isolated from non-selective agar, and the erythromycin resistance of the isolates was subsequently tested by velvet replication. The large majority of the isolates proved to have, as expected, the recipient ery-r2 phenotype: these replicated to agar containing 0.25 but not 10 or 100 pg/ml erythromycin. Twenty-seven isolates, on the other hand, replicated normally on 0.25 pg/ml erythromycin but also threw off from a few to many papillae on 10 and 100 pg/ml erythromycin. These isolates, therefore, appeared to be genotypically ery-r2-r8/+. A papilla growing on 100 pg/ml erythromycin was removed in the case of three such isolates. Cultures made from these papillae proved to be stably resistant to 100 pg/ml erythromycin. Their presumptive genotype was ery-r2-r8.

A DNA preparation was made from each of these presumptively ery-r2-r8 cultures and then tested on wild-type sensitive cells. The results are summarized in Table 8. In brief, it may be stated that each of these preparations produced three classes of transformants: ery-r2, ery-r8/+, and ery-r2-r8/+. These classes were identified not only by their phenotypes but also by the types of segregants they produced. Since both the ery-r2 and ery-r8 markers were thus shown to be present in the DNA preparations, the genotypes of the donors were thereby confirmed. To determine the degree of linkage, if any, between the my-7-2 and ery- r8 markers in these experiments, a sample of transformed colonies selected on 0.25 pg/ml erythromycin was isolated and tested in the case of one of the DNA preparations (# 1 ) , which had been used at both a barely saturating (1 pg/ml) and a limiting (0.01 pg/ml) concentration. At the limiting concentration the

TABLE 8

Transformiion of wild-type Challis cells by DNA from ery-r2-r8 donors

Transformants/ml resistant to Erythromycin

Streptomycin 0.25 pg/ml 100 pg/ml

Donor DNA (pg/ml) ( s t r d 3 ) (ery-r8/+) (erpr.2) ( e ~ - r z - r 8 / + ) err-rz-rs) DNA conc. 5000 pg/ml 0.05 pg/ml seg- Sege (ery-r8 or

str-r43 ery-r2 1 3.8 x 10" 0 3.7 x 105 0 0 str-r43 ery-rg 1 3.8 x 105 8.2 x 10s 0 0 6.4 x 103 ery-r2-r8 #I 1 0 3.2 x 10s 3.2 x 105 2.6 x 104 <IW

0.01 0 NC 5.6 x 103 2.0 x 1012 <io2

ery-r2-r8 #Z 1 0 3.7 x 105 2.7 x 105 5.5 x 103 <I@ ery-rZ-r-8 #3 1 0 2.2 x 105 2.2 x 105 2.2 x 103 <io3

* seg and segf = transformants incapable or capable, respectively, of throwing off segregants that can grow on 100 @ g / d erythromycin. Figures in seg+ column are calculated from the proportion of colonies selected in 0.25 pg/ml erythromycin that subsequently yielded segregants. The actual proportions were 8/98, 8/223, 2/98 and 1/98 for the falloiwing DNA preparations, respectively: #I at 1 @g/ml, #I at 0.01 pg/ml, #2 at 1 pg/ml, and #3 at I p g / d DNA.

NC = not checked.


frequency of transformation was nearly two orders of magnitude lower than the frequency obtained with the saturating concentration. The samples were tested to determine the proportion of ery-r2 transformants also containing the ery-r8 marker. This proved to be 8% for the sample obtained at high DNA concentration and 4% for the sample at low DNA concentration. The two ery-r markers are obviously linked, although the probability of separating them from the donor molecule containing them is relatively high (92-96%).

Transformations involving ery-r8 as a recipient marker: A stably transformed ery-r8 strain which was also s t r f was rendered competent to undergo transformation by: (1) DNA ery 4- str-r43 and (2) DNA ery-r-2 str-r43. The transformed cultures were plated in non-selective media after termination of exposure to 1 pg/ml DNA, and two hours later an overlay was made with either non-selective or streptomycin-containing medium. As may be seen in Table 9, the ery-rg 4- strain was highly competent, as indicated by the high efficiency of str-r4? transformations. In these experiments. a large number of colonies was isolated from the non-selective plates in the expectation that, upon test for level of resistance to erythromycin, some might prove to be transformed at the ery locus.

In the case in which the donor DNA was ery + str-r43, one would expect ery + transformants if the ery-r8 recipient allele were simply replaced by its wild-type homolog. If the frequency of this replacement were at least equal to that of str-r transformations, a total of 19 ery 4- transformants should have been found among the 136 isolates tested. On the other hand, if the ery + donor allele were simply added on to the ery-rg recipient allele to form a merodiploid, a total of 36 ery- 7.8/+ merodiploids should have been found. Neither ery + nor ery-r8/+ transformants were found, however. All of the isolates were ery-r8 in phenotype.

TABLE 9

Stability of ery-rg as a recipient marker

No. colonies Competent isolated f"

Donor population Efficiency non-selective DNA no. str-r transf. media

ery + str-r43 1 0.22 57 2 0.08 79

136 ery-r2 s t r 4 3 1 0.26 275

No. ery t

No. ery-rZ

No. ery-r8/+

Exp. Obs.

13 0 6 0

19 0 0 0

~~

Exp. Obs.

0 0 0 0

0 0 4 0

- -

Exp. Obs.

24 0 12 0

36 0 128 0

_____

_ _ _

A transformed Challis strain carrying the ery-r8 and str + markers was exposed to 1 pg/ml of DNA indicated. Efficiency of str-r transformation = no. str-r transformed colonies arising in agar after streptomycin overlay/no. colo!nies appearing in similar medium not receiving a streptomycin overlay. Exp. = expected; obs. = observed. Exp. no. err-&/+ calculated as: no. colonies isolated x eff. str-r transf. x eff. merodiploid formation (relative to sfr-r trans. in the cross ery + str + X DNA ery-7-8 str-r43; = 1.9). For the case in which ery-r2 str-r43 was used, the exp. no. thus calculated was further multiplied by the relative efficiency 04 exchange between the ery-rz and ery-r8 sites (as determined in the cross ery + str + X DNA ery-r2-r8 str-r43; = 0.94). Exp. no. ery + calculated as: no. of colonies isolated x &E. str-r transformation. Exp. no. ery-r2 = no. colonies isolated x eff. str-r trans x prob. d m-integration of ery-r-2 and wild-type homolog of ery-r8 (= 0.06).


Similarly, in the case in which the donor DNA was ery-rZ-str-r43, separation of the ery-r2 donor allele from the wild-type donor allele homologous to the ery- r8 site should be expected to occur frequently (about 94% of the time, based upon our previous results obtained by crossing wild-type recipients with DNA ery-r2-r8). Assuming the wild-type allele would add on to the ery-r8 allele to form a merodiploid, one should expect a total of 128 merodiploids out of the total of 275 colonies isolated. Yet no merodiploids were found. Nor were any ery-r2 iransformants observed among the isolates. Rather all of the isolates were ery-r8 in phenotype.

The recipient ery locus into which the ery-r8 marker has been integrated thus appears to be refractory to subsequent transformation.

Other spontaneous ery-r mutations like ery-r8: In the Challis strain the frequency of spontaneous mutants selected at concentrations between 0.1 and 0.25 ug/ml erythromycin varied between and per c.f.u. Fifty-five independently arising mutants were isolated in order to determine the relative frequency of mutations with properties like those of ery-r8. Two of these mutants, which were selected in a single step at concentrations b?tween 0.1 and 0.25 pg/ml erythromycin, exhibited the high level of resistance observed in the ery-r8 strain. The majority resisted no more than 1 pg/ml of the antibiotic, and DNA from mutants of this type produced in wild-type recipients transformants selectable with about equal efficiency on 0.05 and 0.25 pg/ml erythromycin. The str-r43 marker was transferred into the two mutants capab!e of resisting 100 pg/ml of the antibiotic (ery-rl6 and ery-r l7) . prior to extracting DNA from each strain. The two transforming preparations were then compared with that from ery-r8 str-r43. The results (Experiment V, Table 2) showed that the ery-rl& and ery-rl7 mutations were very similar to ery-r8: they all induced in wild-type recipients a much higher frequency of transformants selectable on 0.05 than on 0.25 ug/ml erythromycin. Moreover, transformants selected on the lower antibiotic concentration proved to be merodiploids with a low level of resistance, while those selected on the higher concentration were stable and exhibited the donor's high level of resistance. Thus, the ery-r8 mutation is not a unique case, and mutations with similar properties can be re-isolated with relative ease.

DISCUSSION

We have presented evidence to show that the ery-r8 mutation in Streptococcus sanguis is, in its transforming activity, unlike most other spontaneous mutations in this or other transformable species of bacteria: it produces heterozygotes with high efficiency. Most HE markers of spontaneous or induced origin produce stable transformants as the majority type; the transformants produced by the nov-rl0 and str-r4? markers in these experiments, for example, are all stable and genotypically identical to nou-rl0 and str-r4? donor strains, respectively. We have shown that the heterozygous state is replicated with a fairly high degree of fidel- ity, since it segregates out the original recipient or donor types with a frequency of about 2 x lO"/bacterium/generation. The fact that the heterozygote is stable to treatment with acridine orange and thymidine deprivation strongly indicates


that the donor ery-r8 marker is integrated into the recipient chromosome and does not become part of a separate replicon. That the ery-7-8 marker is integrated near its homologous allele is, furthermore, indicated by the finding that limiting concentrations of DNA from the heterozygote exhibit one-fourth the ability to produce the heterozygous state in wild-type recipients as do similar concentrations of DNA from stable ery-7-8 mutants. The likeliest explanation for this observation is that single molecules of DNA extracted from heterozygotes carry both the original recipient (+) and donor ( r8 ) alleles, the ery-r-8 marker having been inserted into the chromosome adjacent to its homologous site. When the heterozygous diploid region is introduced into a wild-type recipient, the only integra- tions capable of yielding an ery-r8/+ genome are substitutions of the entire donor region for the haploid homolog or additions of the donated ery-7-8 marker only, neither of which are expected to occur with high efficiency. We postulate, moreover, that the diploid condition is intrinsically unstable, throwing off with about equal frequency either the original donor o r recipient allele by looping out of a segment carrying one allele or the other followed by recombination (RAVIN and TAKAHASHI, 1970). The hypothesis we propose is entirely consonant with the results obtained by introducing the ery-r8 marker into a strain carrying the ery-2 mutation, a typical HE marker. In this case the predominant transformant is genotypically ery-r2-r8/+; i.e., one in which the ery-r2 marker is not replaced and the ery-r8 marker is added on to its wild type allele. The 18 marker cannot be far from the ery-r2 mutated site, which is known to affect ribosomal resistance to erythromycin, since single DNA molecules from ery-r2-r8 segregants can transfer both markers. The significant fact is that heterozygosity is not introduced at the ery-7-2 site when the ery-r-8 marker establishes a heterozygous diploid region upon integration. The region of duplication is short. Hence. we speak of the ery-r8 marker as producing merodiploids.

We have considered alternative explanations and found them deficient fo r various reasons: ( 1 ) One might suppose that the ery-r8 strain contains four unlinked plasmids carrying an identical erythromycin-resistance mutation, while the ery-r8/+ strain carries only one such plasmid, in order to account for the difference in relative transforming activity of DNA from the ery-r-8 and ery-r8/+ strains as well as for the dependence of ery-r8 transformant production upon multiple DNA infection. In this case, however, it would be difficult to explain the extreme stability of the resistance of the ery-r8 strain under a wide variety of conditions and the relative ease of obtaining spontaneous mutants similar to ery-rg. (2) Alternatively, one might suppose that there are four separate but identical eryf loci in the wild-type Challis chromosome, that only one of these loci is transformed by a single molecule carrying the ery-r8 marker, and that subsequent “segregation” of a stable ery-rg cell consists in the rapid conversion of these loci to a homozygous mutant condition. The relatively high frequency of spontaneous mutations with properties like those of ery-r-8 would also have to be explained, on this alternative view, as due to a rapid spread of the mutated state from the initially affected locus to all four loci. These ad hoc assumptions strike us as unlikely. ( 3 ) Indeed, the same objectionable assumptions would have to be


made even if one supposed the replicate loci were closely linked, as in a tandem repeat. Moreover, were the stable ery-r-8 mutation a homozygous mutated repeat, it is not clear why, upon transfer, such a mutation could not be stably substituted for its allele, the homozygous unmutatd repeat, rather than having to dissociate so that only a single mutated segment could substitute at a time for a single unmutated segment in the recipient. In short, the suppsition that diploidy or polyploidy of the ery locus exists stably prior to transformation creates more problems than it solves.

Although the ery-7-23 marker produces merodiploids with high efficiency during transformation, stable transformants phenotypically and genotypically like the donor strain do appear at low frequency. The origin of these stable transformants is probably the segregation of the wild-type alleles from a small proportion of the originally induced merodiploids. Support for this view comes from several direc- lions: (1 ) the production of stable ery-r8 transformants, when induced by single molecules of DNA, is no more sensitive to ultraviolet light than the production of merodiploids. The production of stable transformants would be highly UV-sensitive if it took place by a process common to LE markers. ( 2 ) Hex- strains of S. pneumoniae which fails to discriminate against LE markers continue to produce stable ery-r8 transformants with low efficiency and merodiploids with high efficiency. ( 3 ) The frequency of stable transformants detected 3 hours after exposure to transforming DNA is about what one would expect from the calculated rate of segregation of ery-rg cells and the number of generations known to elapse in this period of time. (4) Rifampin treatment of recipient cells has a strong enhancing effect on the production of stable transformants, and it has a definitely stimulating effect on segregation from merodiploids. The effect of RIF in transformation has often been greater than what we have observed in segregation, however. This may be due to the fact that cells competent to undergo transformation are in a distinct physiological state, which may be somewhat conducive to segregation.

The question that remains concerns the nature of the ery-r8 mutation. We do not see any unequivocal way of deducing the physical nature of the ery-r8 marker from our experiments. However, several significant facts should be borne in mind in any interpretation of this marker: (1) It is not uncommon among spontaneously occurring mutations. At least among spontaneous mutants selected at a low concentration of erythromycin. mutations with properties like those of ery-7-8 appear with the frequency higher than 1%. (2) Mole- cules carrying the ery-r8 marker cooperate in the production of stable transformants. Thus, recipients receiving more than one molecule of DNA bearing ery-7-8 yield at least seven times more stable transformants than do recipients receiving only one. The nature of this cooperation is not understood, although it undoubtedly reflects the physical state of the ery-r8 region. Possibly the presence of a second molecule of DNA bearing ery-r8 increases the chance of segregation by a recipient that has already become an ery-r8/f merodiploid. Whatever be the mechanism by which inter-molecular cooperativity occurs in producing stable ery-7-8 transformants, it is clear that such cooperativity is inhibited by prior dam-


ages induced by ultraviolet light. ( 3 ) Once stably integrated into the recipient genome, the region containing the ery-r8 marker is difficult to transform. With neither wild-type DNA nor ery-r2 DNA (which is known to contain the wild- type homolog of ery-r8) can ery-r8 recipients be as easily converted to merodiploids as wild-type or ery-r2 recipients can with ery-r8 DNA. Hence, ery-7-8 is a marker that does not transform with equal efficiency in reciprocal crosses. Since the production of a merodiploid must be the colnsequence of an illegitimate or “unequal” crossover, this is tantamount to saying that unequal crossing over does not have the same likelihood in both directions of the cross.

It is our supposition that the ery-r8 mutation is some gross “chromosomal” aberration, such as an inversion o r large deletion in the sequence of DNA bases in the ery region of the genome. This supposition is not yet susceptible to easy direct confirmation. Although we are left in an inconclusive state with regard to the nature of the ery-7-8 mutation, its study has provided some important caveats for students of transformation. Firstly, this study warns future (and past) investigators that certain markers may arise that may oaly appear to be LE markers. If such markers produced at high efficiency merodiploids that are phenotypically indistinguishable from the wild-type recipients, the true HE nature of the marker might never become known. It is advisable to test all ap- parent LE markers under the least stringently selective conditions in order to allow for the detection of merodiploids. if they occur at all. In this regard, we have recently re-examined the ery-rl0 marker of S. pneumoniae which in many respects resembles the e r y d marker of S. sanguis. It was previously found to produce merodiploids in ery-r2 recipients (RAVIN and TAKAHASHI 1970). When we transformed wild-type recipients with ery-riO DNA and selected at the lowest concentration of erythromycin that would suppress wild-type growth, we failed to uncover any merodiploids of low resistance; all of the transformants, which occurred at low frequency, were identical to the donor. This may mean only that the ery-rlO/+ phenotype is impossible to distinguish from the wild phenotype. Secondly, when the transforming activity of a mutant donor DNA appears to increas? as the square of the DNA concentration, over a certain range of concentrations, the finding cannot alone be taken as proof that two independently transferred markers are responsible for the mutant phenotype. We have seen in our studies the case of stable integration enhanced by cooperation between two molecules of DNA bearing the same mutation.

This research was supported at different times by grants from the National Science Founda- tion (GB-18511) and the National Institute for Allergy and Infectious Diseases (AI-09117), for which the authors are deeply grateful.

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a transforming marker that produces …replicon by donor dna that has failed to become physically...

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