Competence Mutant of Haemophilus influenzae Transformation - NCBI

JOURNAL

OF

Vol. 112, No. 1 Printed in U.S.A.

BACTERIOLOGY, Oct. 1972, p. 492-502

Copyright © 1972 American Society for Microbiology

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation JOHN H. CASTER' AND SOL H. GOODGAL Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication 26 April 1972

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com-56, was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process. In bacterial transformation, free deoxyribonucleic acid (DNA) molecules are absorbed by physiologically competent cells, which can be shown to have acquired new genetic characteristics as a consequence. An understanding of this phenomenon is of importance. Transformation is the simplest known form of sexuality, the prototype for possible correction of human genetic defects and one of the most accessible systems for the study of recombination mechanisms. As an approach toward understanding the processes involved, we have isolated and characterized Haemophilus influenzae mutants unable to transform with normal efficiencies under standard conditions (J. H. Caster, E. H. Postel, N. Stanton, and S. H. Goodgal, Bacteriol. Proc., 1969, p. 60, and I Present address: Department of Microbiology and the School of Medicine, Southern Illinois University, Carbondale, Ill. 62901. 492

reference 5). Such com- strains exhibit a variety of phenotypes and contain material for the study of various aspects of the transformation process. We have given our attention first to an investigation of those mutants deficient in yield of transformants despite adequate DNA uptake (class 3 mutants, reference 5). Surveys of the fates of donor DNA and of radiation sensitivity in such strains have previously been published (20, 21). Among the class 3 mutants were several which were transformed to certain characteristics with widely different frequencies. Genetic markers incorporated with no more than twofold differences in efficiency by the normal com+ strain would be incorporated with 10fold or even greater differences in efficiency by these particular strains. We designated this phenomenon, without prejudication, the "ratio-effect," and report here our findings

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from a study of one such strain. A preliminary report was presented previously (J. H. Caster, E. H. Postel, N. Stanton, and S. H. Goodgal, Bacteriol. Proc., 1971, p. 34). MATERIALS AND METHODS Bacterial strains. Strains used were derived from H. influenzae Rd (1) which was the com+ strain used throughout the course of these studies. Antibioticresistant strains have been described (17) (but see below). Transformation-deficient strain comr56 was isolated from an Rd stock by its failure to transform to streptomycin resistance when replica-plated onto a plate spread with str-marked DNA after Nmethyl-N-nitroso-N'-nitroguanidine treatment (5). The particular Rd stock from which comr56 was derived did not adsorb S2 bacteriophage. To eliminate this characteristic, as well as to reduce the possibility that the various characteristics of com-56 were due to several distinct mutations, the com-56 characteristic was transformed into a phage-sensitive Rd stock and then designated com-56Rd. (Transformants were detected by the plate transformation procedure above.) Except for phage sensitivity, com56Rd behaves essentially as the original com-56 strain and was substituted for it in some experiments. Antibiotic-resistant strains were derived from com-56 and com-56Rd by transforming the strain with suitably marked DNA and by testing isolated transformants to insure that they still retained the com- character. Three other strains isolated by us are similar to com-56, com-46, com-47, and com-48 are thought to be three separate isolations of the same mutation. They were obtained in the same trial and cannot be separated by recombination. They are closely linked to com-56, but can undergo recombination with it. Physiologically, they appear identical to com-56 and probably represent a lesion in a different part of the same gene.

S2 phage was isolated from a strain of H. influenzae from a patient at the Hospital of the University of Pennsylvania (3). Nomenclature of novobiocin resistance. The designations of the novobiocin-resistant marker have undergone an unfortunately complex history. The original mutation, which confers resistance of 5 lAg of novobiocin (Cathomycin) per ml, but which is usually selected for at a 2 or 2.5 Ag/ml concentration of the antibiotic, was isolated by Goodgal (10), who called it C, and later nour2½/2 (25). Day and Rupert, who have analyzed this region, designate the same marker Nb1 (8). A strain resistant to 25 gg or more of novobiocin per ml was isolated by Voll from the lower-level resistant strain (M. J. Voll, Ph.D. thesis, Univ. of Pennsylvania, 1964). This has been referred to as C21, novr25, or simply noUr by this laboratory, and Nb2 by Day and Rupert (8), who have shown that the high-level resistance is conferred by the presence of Nb1 and a second, closely linked mutation which they designate Nb,. Nb8, by itself, confers resistance to only 0.5 to 1.0 gg of the antibiotic per ml.

493

We will utilize a new nomenclature which better conforms to modern practice (9). We will designate the Nb, locus of Day and Rupert as novA and the Nb3 locus novB. The double mutant, designated Nb2 by Day and Rupert, will be referred to here as novAB. The phenotype of cells containing only novA will be abbreviated "Lo-Nov-r." The phenotype of cells containing the double mutant novAB will be abbreviated "Hi-Nov-r." (The very low-level-resistant phenotype of novB alone will not be utilized.) In the experiments reported here, all novobiocin-resistant donor DNA was novAB. Presence of the double novAB marker was determined by selection at a 20 ,g/ml final concentration of the antibiotic. In some experiments, we sought to determine the degree of linkage between novA and novB. Cells transformed with novAB DNA, able to grow at a concentration of 2 Mg of novobiocin per ml, but not at 20 Mg/ml, were assumed to have integrated only the novA site. Culture media and conditions of growth. Experimental conditions were as described by Goodgal and Herriott (11), unless otherwise indicated. Standard growth medium was brain heart infusion (BRI; Difco) supplemented with hemin and nicotinamide adenine dinucleotide (NAD) (17). Defined growth medium M-Ic was described previously (15). Competence procedures used were as follows: standard procedure, the Cameron modification (2) of the Goodgal and Herriott procedure; extended procedure, modified standard procedure with a 90-min period of incubation without shaking (nonaerobic) followed by 60 min of shaking; and two-stage M-IV procedure, as given by Herriott, Meyer, and Vogt (14), using heart infusion in the first stage and 130 min in M-IV in the second stage. 32P-labeled DNA was prepared from cells grown in M-Ic medium without P04, except for 0.5 to 1.2 mCi of carrier-free P2p as orthophosphate, and the small amount of phosphate added with the inoculum (6 x 107 cells in 0.02 ml of BHI into 20 ml of M-Ic medium). A 125 to 250-ml Erlenmeyer flask containing the culture was held at an angle of about 500 from the vertical and rotated slowly (2 to 4 rev/min) on its main axis in a 37 C room overnight. This degree of aeration appears to be optimum (J. Michalka, Ph.D. thesis, Univ. of Pennsylvania, 1970). Maximum cell density obtained was about 4 x 108 cells/ml. 3H-DNA was prepared by the same technique, except that M-Ic complete with phosphate was used, 3H-thymidine was added after the culture had become visibly turbid (about 108 to 2 x 108 cells/ml), and growth was stopped when the culture reached 4 x 1011 cells/ml. Heavy 32P-DNA was prepared from cells grown in the medium of Goodgal and Postel (12), without phosphate, and with half of the D20 replaced with H20. Otherwise, growth conditions were the same as for normal density 32P-DNA. 3H-labeled competent cells were prepared by the standard or extended procedure, with 3H-thymidine added when the density of the culture in logarithmic growth had reached about 2 x 108 to 3 x 108

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cells/ml. Preparation of DNA. Genetically marked DNA for transformations was prepared as previously described (11), except that cells were grown in BHI with hemin and NAD and were lysed by the addition of sodium dodecyl sulfate (SDS) to a final concentration of 1%. Small amounts of high-specific-activity isotopelabeled DNA and high-molecular-weight DNA, were prepared by the method of Michalka (Ph.D. thesis, 1970). About 8 x 109 labeled cells were washed two to three times in SSC (0.15 M NaCl, 0.015 M sodium citrate) and resuspended in 0.5 ml of an ethylenediaminetetraacetate (EDTA)-NaCl solution (6 x 10-4 M EDTA and 0.01 M NaCl, pH 7). A 0.1-ml portion of a solution of lysozyme (2 mg/ml) was added and mixed, and the suspension was incubated for 2 hr at 37 C without shaking. SDS (0.1 ml of a 10% solution) and then Pronase (0.1 ml of a 10 mg/ml solution) were added gently. The lysing solutions were allowed to flow down the side of the tube and were mixed by tilting slowly to near horizontal and then returning to an upright position. This lysate was incubated overnight at 37 C without shaking. Lysates prepared in this manner contain cellular DNA in predominantly high-molecular-weight form. Further purification could be satisfactorily accomplished by carefully layering 0.2 to 0.3 ml of such lysates in a wide-bore 1-ml pipette on the top of 4.5 ml each of 5% to 20% sucrose gradients (1 M NaCl, 0.015 M sodium citrate, pH adjusted to 10.5 with NaOH). The gradients were centrifuged in an SW39 rotor for 2 hr at 29,000 to 32,000 rev/min in a Beckman ultracentrifuge. After centrifugation, 30 to 35 equal volume fractions were collected by positive displacement through a hole in the bottom of the tube. The material collected in the region of fractions 10 through 20 contained predominantly DNA. In the case of 32P-labeled lysates, this was followed by a more slowly sedimenting but more intensely radioactive material containing the bulk of the ribonucleic acid. 32P-labeled DNA was prepared individually for each experiment and used within 1 to 2 days in order to obtain the greatest specific activity with the least biological inactivation. Lysates from transformed cells were prepared by the same general procedure, but were not sedimented in sucrose except where this was required for their analysis. In certain early experiments, the Notani and Goodgal (19) modification of the method of Frankel (10) was used. DNA size was preserved, where required, by very slow pipetting with large-bore pipettes (17; Michalka, Ph.D. thesis, 1970). Intentional shearing of high-molecular-weight DNA was accomplished by repeated passage through a 26 gauge needle. Phage S2 DNA was prepared by the method of Bendler (J. Bendler, Ph.D. thesis, Johns Hopkins Univ., 1968). DNA binding determination. DNA binding and transformation efficiencies were determined as previously described (5). Determination of genetic linkage. As used in

J. BACTERIOL.

this paper, genetic linkage refers to the percentage of transformant clones, selected for one marker, which subsequently can be shown also to be transformants for a second marker. Such transformations were done at limiting DNA concentrations, and appropriate controls were used to correct for spontaneous mutations. Usually the transformant clones were picked with sterile toothpicks and stabbed into agar selective for the second marker. In some cases replica plating was used, but it had the disadvantage that H. influenzae does not grow as reproducibly on the surface as it does within the agar. Transfection. Transfection techniques were essentially those of Harm and Rupert (13) and Bendler (Ph.D. thesis, 1968). A 0.1-ml amount of phage DNA and 0.1 ml of competent cells were mixed in 2.3 ml of BHI and incubated for 20 to 30 min at 34 C with shaking. Deoxyribonuclease was then usually added and incubated for 5 to 10 min at 37 C. BHI agar containing 3 x 10-4 M Ca2+ and 1.5 x 10-4 M Mg2+ (2.5 ml) at about 60 C was pipetted into the transformation mixture, and 2 ml of the combination was quickly spread on the surface of each of two BHI agar plates (with added Ca and Mg). In some cases, the transformation mixture was diluted 1:10 before addition of agar. Where bacterial markers were to be assayed, samples were removed for this purpose before addition of agar. No indicator cells were added, as nontransformed cells in the mixture serve this function. Analysis of donor activity in transformed cells. The kinetics of recoverability of donor and recombinant (linked donor-recipient) marker activity was determined by the method of Voll and Goodgal (25). Lysates were made by the method of Notani and Goodgal (19). Control experiments assured that lysates were assayed under conditions of limiting DNA concentrations and permitted correction for spontaneous mutations. (It is important to correct for spontaneous mutations occurring within the DNA of the recipient cells from which lysates are made, as well as those which occur in the cells used to assay the lysates.) Relative frequencies of novAB (the donor marker) and novA alone were determined by testing low-level transformant clones for their ability to grow at high levels of the antibiotic, as well as by selecting for transformants at both low and high levels initially. Physical association of donor and recipient material was determined by the sucrose gradient method of Notani, Frankel, and Goodgal (18). In a typical experiment, str and str comS56 cells were each labeled with 1 mCi of 3H-thymidine (in 2.5 ml) and made competent by the extended procedure. Cells (0.2 ml each) were diluted into 5.8 ml of BHI (without hemin or NAD), containing about 0.03 Ag of sheared 32P-labeled novAB DNA per ml, and incubated for 10 min at 34 C with shaking. Hemin, NAD, and 240 ug of deoxyribonuclease with MgCl2 (final concentration, 3 x 10- M) were added (zero time), and the temperature was raised to 37 C. Samples were removed at various times and diluted and plated appropriately for determination of total viable centers and transformants, or diluted into an

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equal volume (2.5 ml) of chilled SSC to be lysed. Lysates were prepared by the method of Michalka (Ph.D. thesis, 1970), taking care to maintain size, and were layered (0.2 ml) onto a 4.5-ml sucrose gradient (5 to 20%, pH 10.5, 0.015 M sodium citrate, 1 M NaCl) and centrifuged at 32,000 rev/min in a Spinco SW39 rotor for 120 min at 20 C. About 38 equal volume fractions were collected alternately on filter paper discs and into CHCl,-saturated BHI broth without hemin or NAD. The filter paper discs were dried, precipitated with trichloroacetic acid, washed twice with acetone, redried, placed in vials with scintillation fluid (17), and counted in a Packard Tri-Carb scintillation counter. BHI samples were warmed to eliminate CHC1I, and then 0.1 ml of competent Rd cells was added to each, incubated for 30 min at 34 C, and diluted and plated to determine the transforming activity of the lysates. Lysates for cesium chloride analysis were prepared similarly. strRd and str com-56 cells were made competent by the M-IV procedure, with 3Hthymidine added during the first stage when the culture reached about 2 x 10' to 4 x 10' cells/ml, as determined by optical density. These competent cells (1 x 109 cells/ml) in 34 ml of M-IV medium were transformed with sheared "2P-labeled novAB DNA containing 2H and "N heavy isotopes (final DNA concentration, 0.006 ug/ml). After 5 min of uptake at 37 C, deoxyribonuclease (20 to 50 ,g/ml) in MgCl was added along with BHI stock (final concentration, 0.9%), with hemin and NAD. Samples were removed at various times for dilution and plating and for preparation of lysates. The latter cells were concentrated 10-fold, yielding about 10 ,g of DNA per ml in each lysate. About 4 ug each of these crude DNA species were sheared and mixed separately with 4.52 g of CsCl in 5-ml volumes, of which 4.5 ml was then transferred to cellulose nitrate tubes (5/16 by 3 inches). Paraffin oil was added to fill the tubes, which were then centrifuged for 3 days at 19 C in a Spinco type-40 angle head at 38,000 rev/min. About 60 equal volume fractions were collected into tubes containing 0.4 ml of nutrient broth. The acid-insoluble radioactivity of a sample of each fraction was determined on filter paper discs, as before. The fractions containing presumptive hybrid material were then pooled, resheared, recentrifuged, and collected as before. Radioactivity and transformation activity of the factions were determined. Controls indicated that the position of the heavy donor DNA was shifted 10 fractions, relative to the recipient DNA, under these conditions. RESULTS

Ratio effect. Table 1 presents the widely differing frequencies with which the ratio effect strain com-56 transformed to resistance to four different antibiotics. The com+ strain Rd transformed to streptovaricin or naladixic acid resistance about twice as often as it did to streptomycin or to high level novAB novobiocin resistance, but the mutant strain trans-

TABLE 1. Numbers of transformants to resistance to various antibioticsa No. of transformants/ml

Strain

com+ com -56

Streptomycin (250)b

Novobiocin (25)

1.8 x 105 1.9 5 x 102

x

Strepto- Nalidixic varicin acid (3) ai 3 (8)

105 3.9 x 105 3.0 x 10'

6 x 10'

3 x 103 1.8 x 10'

aThe standard procedure for preparing competent cells was used for both com+ and com-56. Total viable counts: approximately 1 x 10' colony-forming units/ml, DNA. b Numbers in parentheses represent antibiotic concentrations (micrograms/milliliter).

formed to naladixic acid resistance as often as 30 to 40 times more frequently than to streptomycin resistance. Transformation not limited by DNA binding. The experiment in Table 2 demonstrates that com-56 bound as much or more DNA as the com+ control, even without preparation by a competence procedure. com-56 showed only minimal response to competence procedures in any case; transformation frequencies were near maximal for exponentially grown cultures. Whereas the frequency of transformation to str-r was similar for com-56 and exponentially grown com+ cultures, other experiments have shown that the ratio effect is characteristic of the strain and is not influenced by the growth regime. As a consequence, transformants to naladixic acid resistance occur significantly more frequently in an exponentially grown culture of com-56 than in an exponentially grown com+ strain. Also, DNA uptake is continuous in com-56, and the number of transformants increases with time of exposure far beyond the time when com+ cells have lost competence, unless uptake is stopped by dilution or deoxyribonuclease treatment. Decrease in genetic linkage. We postulated that the ratio effect might reflect a general reduction in genetic linkage, since the str and novAB markers are known to involve multiple lesions, which must be incorporated together to convey full resistance. In agreement with this, when cells were transformed with DNA containing the novAB marker, and then challenged with 2.5 ,g of novobiocin per ml (to which resistance is acquired by the incorporation of novA alone), it was found that these Lo-Nov-r transformants arose with a frequency about 10-fold greater than that for resistance to higher levels of the antibiotic. In a somewhat more rigorous experiment (Table 3, experiment A), cells were transformed with

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TABLE 2. Deoxyribonuclease-resistant 32P-DNA binding versus yield of str-r transformants by com + and com -56a Prepn

com + Standard competence proce- 3 dure . Exponential growth .5 com -56 Standard competence proce- 5 dure . Exponential growth .2

str-r

Counts per min per ml

x 106

548

x 103

30

x 103

1,232

x 103

747

a Cells were prepared by standard procedure or by direct exponential growth to the same density. Total viable counts: approximately 1 x 109 colony-forming units (CFU)/ml.

novAB DNA, and transformants resistant to the lower level of the antibiotic (those incorporating at least the novA locus) were individually picked and tested to determine how many had also acquired resistance to the higher level (and thus could be assumed to have incorporated novB as well). The standard agar overlay procedure which was used does not permit division of transformants. Whereas 96% of these com+ colonies showing any novobiocin resistance were resistant to high levels (and thus incorporated both novA and novB), only 12% of the resistant colonies from com-56 appeared to have acquired both sites. The remainder showed the novA low-level resistance, but did not grow when tested at 20 ,g of the antibiotic per ml. Although transformation frequencies are low in com-56, appropriate controls indicated that less than 20% of the Lo-Nov-r cells could have arisen by spontaneous mutation. In another experiment (Table 3, experiment B), cells transformed with doubly marked high-molecular-weight str novAB DNA were selected for streptomycin-resistant transformants. These were then picked and tested to determine how many were also resistant to novobiocin. Whereas 48% of the com+ str transformants were also novAB, only 7% of the str com-56 transformants were resistant to high levels of novobiocin. (An additional few, about 1%, were resistant only to low levels of novobiocin. These may include some spontaneous mutations. Spontaneous mutations to str or novAB occur so infrequently that they make no significant contribution to these classes even at the low transformation frequencies characteristic of com-56.) Other experiments (not presented here) have

also indicated reduced genetic linkage in com56, consistent with our hypothesis. Integrity of donor activity. A variety of events could reduce genetic linkage. To distinguish among the various possibilities, we first asked whether the donor genetic material was in any way inactivated before its association with the recipient chromosome. Preliminary experiments (see reference 20) had indicated that donor DNA was not reduced to acid-soluble form to any greater extent than in com+ strains, but less obvious damage to the genetic material might be sufficient to destroy a proportion of its information content and thereby reduce the apparent genetic linkage between donor markers. When competent cells are treated with DNA extracted from an appropriate bacteriophage, lytic cycles are initiated in a small fraction of the cells. This process of transfection was expected to be especially sensitive to donor DNA degradation, since damage to almost any part of the genome would be expected to block a lytic infection. Contrary to this expectation, Table 4 shows that com-56 transfected fairly efficiently with phage S2 DNA, producing about 10% as many plaques as the com+ control. Unlike the com+ strain, however, com-56 failed to transfect detectably with prophage DNA (DNA extracted from a lysogenic bacterial strain) (Murray, Caster, and Goodgal, 1972, manuscript in preparation). TABLE 3. Linkage between markers Strain

com + com-56

Expt Aa

Expt Bb

0.96 0.12

0.48 0.07

aFraction of transformants selected on 2.5 jug of novobiocin per ml that were also resistant to 25 Ag of novobiocin per ml. bFraction of transformants selected on streptomycin that were also resistant to 25 jAg of novobiocin per ml. TABLE 4. Transfection of comr+ or com -56 cells by S2 phage DNA PFU/mla Phage DNA concn (jAg/2.5 ml)

com+

com -56

0.01 0.1 1.0

1,993 6,825 > 10,000

461 1,236

156

a Average of four tests; PFU, plaque-forming units.

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Another test for the possibility of donor degradation was to measure donor marker activity recoverable fro] lysates of transformed cells. Streptomycin-: resistant com+Rd or com-56 cells were trans3formed with novAB DNA. Lysates were mac Se at various times after DNA uptake, and usEad in turn to transform Rd cells sensitive to b oth antibiotics. As previously reported (25), novAB activity in com+ cells decreased initiaally and then increased (in parallel to the in icrease in recipient str transforming activit ;y, not shown), whereas linked str novAB actiivity increased rapidly at first and later at a rate similar to the increase in novAB alone, vvhich presumably reflects chromosomal repliccation (Fig. 1). Donor marker activity in lys&ates of com-56 fell somewhat m

more drastically than in com+, and, unlike the latter, did not increase. However, the initial level of activity was higher, due to greater DNA uptake, and the loss of activity was not sufficient to account for the low frequency of transformation observed. More significantly, novAB and novA activities were lost at the same rate, indicating that the inactivation of donor activity seen here was not the source of the ratio effect. On the other hand, no linked nov str activity could be detected above the level of spontaneous mutation. We concluded that although some loss of

donor marker activity occurred, this did not account for the low transformation rates

widely differing marker efficiencies this strain.

or

seen

the in

Association of donor material with the chromosome. The absence of detectable linkage between donor and recipient markers in lysates of com-56 suggested that donor DNA might not associate with the recipient chromosome in normal amounts. Perhaps the bulk of the donor DNA did not even penetrate into the interior of the cell, but remained sequestered in some deoxyribonuclease-protected area outside the membrane.

4

To test this possibility, str com+ or com-56 cells labeled with tritiated thymidine were

LL

transformed with 32P-labeled novAB DNA.

-s--'

..

made at various times after DNA care to avoid shearing of the recipient DNA. These lysates were then layLysates

0

0

were

uptake, taking

0

ered onto sucrose gradients and sedimented. Fractions were assayed for radioisotopes and donor and recipient marker activity. Figure 2a shows that by 30 min after addition of DNA there were detectable amounts of both donor radioactivity and transforming activity in the high-molecular-weight region of Rd lysates, although transformants were not proportional to radioactivity in this region.

L 0

30

60

90

mil FIG. 1. Donor X and recombinant marker activity in lysates from tran sformed cultures of com+ or com[f

56 as a function o time after DNA uptake. (The first sample [t=O mir f] was taken after 5 min of DNA uptake plus 2 mi,n of deoxyribonuclease treatment.) Ordinate represeints number of antibiotic-resistant cells obtained wihen sensitive Rd cells were transformed with the lysates at a limiting DNA concentration. Curve A, Donor activity in com-56; curve B, donor activity in com+; curve C, recombinant activity in com+. Recombinant activity in com-56 could not be det(ected. Symbols: 0, Lo-Nov-r cells; 0, Hi-Nov-r cells 3. Points on curve C represent cells resistant to strept

tomycin as well as to novobiocin.

com-56 30-min lysates (Fig. 2b) had no detectable donor transforming activity in high-molecular-weight region, despite the the recovery of a substantial portion of the donor 32p from this portion of the gradient. The 60-min lysates (Fig. 2c) were essentially the same. We concluded that com-56 associated approximately normal amounts of donor material with its chromosome, but that this associated material

was

somehow

deficient

in

donor

genetic

activity.

Deficiencies in enetic activit could have guledcfro yncompeteo abnoul hav from incomplete or abnormal integraresulted tion. Experiments to detect non-convalently linked material, abnormally sedimenting tangles, or single-stranded donor activity were

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CASTER AND GOODGAL

Fraction

4._ u

Fraction 1.0

I

10

20 Fraction

30

FIG. 2. Association of donor DNA activity with high-molecular-weight (HMW) donor material. Analysis of lysates of transformed cells by sucrose gradient sedimentation. a, com+ cells after 30 min of incubation; b, com-56 cells after 30 min of incubation; c, com-56 cells after 60 min of incubation. Curves are drawn to a variety of scales. Full-scale activities are indicated below in parentheses.) Recipient: 3H-thymidine (0) (all graphs, 1.0 = 6.9 x 103 counts per min per ml); str transformant activity (A) (graph a, 2.3 x 106 CFU/ml; graph b, 7.2 x 105 CFU/ml; graph c, 5.8 x 106 CFU/ml). Donor: 32P (0) (graph a, 4.6 x 103 counts per min per ml; graph b,

J. BACTERIOL.

without positive result, although none had sufficient discrimination to exclude these possibilities decisively (20). It was clear, however, that the association of donor material with the recipient was not usually lethal (23): the addition of DNA did not significantly lower the viability of com-56 cultures as compared with com+, although the amount of DNA normally absorbed indicates that the majority of the cells participate in the transformation process (Table 2). Activity a reflection of integration size. The deficiency in donor genetic activity in the high-molecular-weight material from comr- 56 lysates could be explained by postulating that this strain incorporated, on the average, shorter pieces of donor DNA than did com+. This would immediately account for the reduction in genetic linkage between markers, and the low efficiency of transformation for multisite markers. It could also explain the moderately low efficiency for transformation of point mutations, provided the assumption was made that shorter pieces are for any reason at a disadvantage in transformation. To test this possibility, DNA was prepared containing the heavy isotopes 2H and '5N, in addition to the radioactive isotope 32p and the novAB genetic marker. com+ or com-56 cultures, labeled with 3H-thymidine and containing the str marker, were transformed with the heavy DNA. Lysates were made from the transformed cells, sheared, and then centrifuged to equilibrium in CsCl. The expectation was that while donor DNA would contribute a measurable density increase to many of the fragments incorporating it in com+ cells, this "hybrid" density material might be significantly reduced or absent in lysates from com-56 since the donor material was postulated to make up a smaller portion of each fragment, on the average. This expectation was fulfilled, as is shown in Fig. 3. Figure 3a shows that donor material extracted from a transformed com+ culture is found associated with light recipient material, but with a substantial shoulder of higher density. Fractions containing presumptive hybrid material were recentrifuged, and this second gradient was tested for both radio- and biological activity, as shown in Fig. 3b. Recipient str and 3H activity are found together. Donor 32p activity has a greater average density than the recip1.7 x 104 counts per min per ml; graph c, 1.7 x 104 counts per min per ml); novAB transformant activity (A) (graph a, 3 x 10' CFU/ml; graph b, 1.5 x 104 CFU/ml; graph c, 1.4 x 104 CFU/ml).

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1' .742=

;

1.74 * ~~~~~~~~~~~~~~1.72

1.72 L. 10 170

4

0

1.70 . ~~~~~~~~~~~~~~~~~~1.68 1.66

1.68 2-7

1\1

-0.1~~~~~~~~~~~~~

4664"siM

0*

1

20 Fraction

40

1

20

40

Fraction

~~~~~~~~~~~~~~~~~~~~~~1.0

1.0

i.76 b

~~~~~~~~~~~~~~~~~1.76 ~~~~~~~~~~~~~~1.740 ~~~~~~~~~~~~~~1.72w . ~~~~~~~~~~~~~~~~1.70 ~~~~~~~~~~~~~~~1.68

-

1.74 1.72 1.70 1.68

0.0

20

40

Fraction

40~~~~~~~~0. OLO1 20

Fraction

FIG. 3. Density distribution of donor and recipient activities in lysates from cells transformed with heavy DNA, as analyzed in CsCI gradients. a, Radioisotope distribution in lysates of transformed com+ cells after 60 min of incubation. b, Radioisotope and genetic marker activity in resedimented presumptive-hybrid fractions from gradient a. Fractions resedimented indicated by "cut" (see graph a). c, Radioisotope distribution in lysates of transformed com-56 cells after 60 min of incubation. d, Radioisotope and genetic marker activity in resedimented presumptive-hybrid fractions from gradient c. (Curves are drawn on a variety of scales. Full scale activities for curves given below in parentheses). Recipient: 3H-thymidine (0) (graph a, 1.0 = 5.0 x 104 counts per min per ml; graph b, 1.8 x 103 counts per min per ml; graph c, 1.0 x 105 counts per min per ml; graph d, 9.7 x 103 counts per min per ml); str transformants (A) (graph b, 1.3 x 106 CFU/mI; graph d, 4.5 x 106 CFU/ml). Donor: 32p (0) (graph a, 5 x 103 counts per min per ml; graph b, 6 x 102 counts per min per ml; graph c, 5.0 x 103 counts per min per ml; graph d, 7.9 x 102 counts per min per ml); novAB transformants (A) (graph b, 2.7 x 103 CFU/ml; graph d, 3.6 x 102 CFU/ml).

ient material, whereas novAB transforming activity is clearly associated with the 32p peak. The denser portion of the 32p peak contains the greater portion of the biological activity, some portion of which may represent unintegrated donor material. In contrast, when lysates of transformed com-56 cells are sedimented (Fig. 3c) there is little if any indication of a hybrid-density shoulder, although there is a small peak of denser material corresponding in density to the donor DNA. The distribution of the remainder of the donor radioactivity simply mirrors the recipient peak.

Fractions from the leading edge of the main peak were recentrifuged and tested for both radioactivity and biological activity (Fig. 3d). The 32p activity is found in nearly the same position as the recipient activity, indicating that there exists no significant number of chromosomal fragments containing sufficient amounts of donor heavy isotopes to cause a detectable shift in density. There is little if any donor transforming activity in this peak. The small amount of novAB activity detected forms a peak located at approximately the same density as the peak of donor activity in com+ lysates. Our conclusion is that most

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donor material incorporated into the chromosome by com-56 is incorporated in pieces significantly smaller in size than are incorporated by com+, and that these small pieces are deficient in their capacity to produce genetic transformation. No evidence for increased single-strand nicks. Our laboratory has previously reported that the ratio-effect strain com-48 is at a disadvantage when transformed with X-irradiated DNA, that is, the number of transformants recovered falls off more rapidly, with increasing X-ray dose, than is true for com+ or for most other com- strains. However, com-48 cell viability itself is no more sensitive to X radiation than the viability of com+ (22). These characteristics are also true of com-56 (Postel, unpublished data). We considered it possible that donor DNA in com-56 might contain an unusual number of single-strand breaks, either because of a hyperactive endonuclease or an inactive repair enzyme, and that this situation would be exaggerated by X radiation. Such nicks might well limit the size of the pieces incorporated into the chromosome. No gross changes in native DNA would be expected from such nicks, but changes in the average size distribution should be detectable. Further, donor strand lengths should be very different after denaturation. Therefore, to examine these possibilities, cold com+ and com-56 cells were transformed with 3H-DNA or 32P-DNA (in both combinations, to eliminate any difference due to the size of the transforming DNA); cells containing different labels were then mixed and lysed together, and the mixed lysates were sedimented on neutral and alkaline sucrose gradients. In no case was the position of the donor peak detectably affected by the cell strain. We therefore conclude that the bulk of the unintegrated donor DNA in com-56 is no smaller, and contains few, if any, more single-stranded nicks than the same DNA in com+. This agrees with the finding of Postel and Goodgal (21) for com-48, although they also report that the size of the unintegrated donor is more sensitive to prior X irradiation in com -48 than in other strains. No evidence for increased breakdown. Incorporation of donor atoms into recipient DNA in smaller than normal-sized pieces and without a concomitant incorporation of donor genes could be explained by digestion of the donor material into nucleotides, which would then be reutilized in repair or replicative synthesis. All evidence presented here and by Postel and Goodgal (20) argues that there is no

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general solubilization of donor material in excess of that occurring in com+ strains. Breakdown of only that portion of the DNA which would otherwise undergo normal integration, with immediate reincorporation into new DNA, might, however, explain the results we have obtained. If such solubilization of donor DNA were responsible for the transformation frequencies characteristic of com-56, we would expect this process to occur even in media incapable of supporting growth, since transformation is known to proceed under such conditions. At the same time, reincorporation of nucleotides might be restricted in nongrowth medium, permitting their detection. To test this, the release of donor radioactivity was tested under conditions similar to those employed by Stuy (24). com+ and com56 cells were made competent by the M-IV procedure, exposed to 3H-labeled DNA for 10 min, centrifuged and washed twice, and then resuspended in saline with 3% Casamino Acids, or M-IV competence (nongrowth) medium. The resuspended cells were maintained at 37 C, and samples were removed at intervals and filtered through membrane filters. The filtrates were collected and dried, and radioactivity was determined in both cells and filtrates. Results were uniformly negative. Both com+ and com-56 release about 50% of their bound radioactivity in 60 min, with similar kinetics, under the conditions given. No increase in solubilization by com-56 can be detected.

DISCUSSION In com-56, atoms from donor DNA are found associated with high-molecular-weight recipient material, but normal donor genetic marker activity cannot be detected in this material. com-56 is therefore placed within the subclass dab- (donor association biologically defective; 20), as are the similar ratio-effect strains com-46, com-47, and com-48. Cesium chloride gradient analysis indicates that fragments of transformed com-56 cell chromosomes contain a smaller density contribution from donor material than do fragments from com+ after transformation with heavy DNA, despite the overall incorporation of similar amounts of material. This result is consistent with either the incorporation of shorter donor strands into a larger number of sites than usual, or excessive degradation of donor DNA, followed by incorporation of the breakdown products during repair or replicative synthesis. These possibilities are not mutually

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exclusive, and the actual situation may reflect a combination of both processes. No general solubilization or excessive cleavage of unintegrated donor material could be detected over the fairly high level of donor material released by com+ strains. Thus, it is very likely that, whatever process is responsible for the reduced average integration size and abnormal transformation efficiencies, this process normally affects only that portion of the donor material associating with the recipient chromosome, during one of the final stages of the integration process. (A considerable solubilization of this specific fraction of the donor DNA might have occurred without detection in our experiments.) Most current models for bacterial recombination envision a pairing between homologous single DNA strands, with removal of redundant or excessive material by endonucleolytic cleavage or exonucleolytic digestion, or both (see reviews 6, 7, 16). Most features of the behavior of com-56 can be explained by postulating excessive endo- or exonucleolytic activity operating on donor material during the integration process. Such activity would account for reduced genetic linkage (and possibly for the ratio effect) and reduced incorporation of point mutations, since donor DNA lost genetic activity below a critical size (Toby Gottfried, Ph.D. thesis, Univ. of Pennsylvania, 1968). If the postulated nuclease activity were to be initiated at nicks in the donor DNA, it would account for the findings that X-irradiated DNA is at a serious disadvantage when used as transforming factor in com-48 and com-56. The fraction of transformants recovered declined more rapidly as a function of dose than in other strains (21). X irradiation would increase the number of single-strand breaks, providing substrates for the enzyme in excess of those provided in normal recombination. Supporting the idea of nuclease activity is the finding that unintegrated, X-irradiated donor DNA (but not unirradiated DNA) is reduced in size in com-48 (21). UV-irradiated DNA, on the other hand, is actually at a lesser disadvantage when transforming these com- strains than when transforming com+ (21). Such seemingly contrary responses to radiation damage become reasonable if one assumes that excision of thymine dimers occurs after integration (22). Excessive nucleolytic cleavage or digestion may well be blocked by the presence of a thymine dimer, thus protecting the genetic information until subsequent integration and repair can occur.

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In any case, defective radiation repair mechanisms are not themselves implicated in the ratio phenomenon. Sensitivity of the cells themselves to either UV or X irradiation is no greater than com+. The recipient chromosome itself appears to be protected from the postulated nuclease. Indirect evidence, therefore, suggests that H. influenzae possesses a donor-specific recombination nuclease, not involved in repair of radiation damage, and that com-56 and the otlier ratio-effect strains contain a genetic lesion which either increases the activity of this nuclease directly or (more likely) removes some normal control upon the extent of its action. It is interesting to correlate this possible hypernuclease activity with the ability of these mutants to take up large amounts of DNA, even though not grown under those specific conditions required for competence production in other strains. A variety of mechanisms can be imagined, but there is nevertheless the strong implication that DNA uptake is somehow related to its metabolism during the integration step. Indeed, it would appear that com-56 and its relatives are, in a sense, hypercompetent; taking up excessive amounts of DNA, but being unable to utilize it efficiently due to the abnormal activity of one part of the integration machinery. In vitro studies of Haemophilus nuclease activity currently in progress may provide direct confirmation for some of these conclusions. ACKNOWLEDGMENTS We acknowledge with thanks the manifold contributions of Nancy Stanton, whose energy, questioning mind, organizational ability, and insistence upon "just one more control" would be inadequately summarized by the usual phrase "excellent technical assistance." This investigation was supported by Public Health Service grant AI-04557 from the National Institute of Allergy and Infectious Diseases. John Caster was a Pennsylvania Plan Scholar.

LITERATURE CITED 1. Alexander, H. E., and G. Leidy. 1953. Induction of streptomycin resistance in sensitive Haemophilus influenzae by extracts containing deoxyribonucleic acid from resistant Haemophilus influenzae. J. Exp. Med.

97:17-31. 2. Barnhart, B. J. and R. M. Herriott. 1963. Penetration of deoxyribonucleic acid into Haemophilus influenzae. Biochim. Biophys. Acta 76:25-39. 3. Bendler, J. W., and S. H. Goodgal. 1968. Prophage S2 mutants in Haemophilus influenzae: a technique for their production and isolation. Science 162:464-465. 4. Carmody, J. M., and R. M. Herriott. 1970. Thymine and thymidine uptake by Haemophilus influenzae and the labeling of deoxyribonucleic acid. J. Bacteriol. 101:525530.

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5. Caster, J. H., E. H. Postel, and S. H. Goodgal. 1970. Competence mutants: isolation of transformation deficient strains of Haemophilus influenzae. Nature (London) 227:515-517. 6. Clark, A. J. 1971. Toward a metabolic interpretation of genetic recombination of E. coli and its phages. Annu. Rev. Microbiol. 25:437-464. 7. Curtiss, Roy III. 1969. Bacterial conjugation. Annu. Rev. Microbiol. 23:69-136. 8. Day, R. S. III, and C. S. Rupert. 1971. Ultraviolet sensitivity of Haemophilus influenzae transforming DNA. I. Effects of genetic mismatch and target size. Mutat. Res. 11:293-311. 9. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-76. 10. Frankel, F. R. 1966. Studies on the nature of replicating DNA in T4-infected Escherichia coli. J. Mol. Biol. 18: 127-143. 11. Goodgal, S. H., and R. M. Herriott. 1961. Studies on transformations of Haemophilus influenzae. I. Competence. J. Gen. Physiol. 44:1201-1227. 12. Goodgal, S. H., and E. H. Postel. 1967. On the mechanism of integration following transformation with single-stranded DNA of Haemophilus influenzae. J. Mol. Biol. 28:261-273. 13. Harm, W., and C. S. Rupert. 1963. Infection of transformable cells of Haemophilus influenzae by bacteriophage and bacteriophage DNA. Z. Vererbungs. 94:336348. 14. Herriott, R. M., E. M. Meyer, and M. Vogt. 1970. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J. Bacteriol. 101:517-524. 15. Herriott, R. M., E. Y. Meyer, M. Vogt, and M. Modon. 1970. Defined medium for growth of Haemophilus influenzae. J. Bacteriol. 101:513-516. 16. Hotchkiss, R. D., and M. Gabor. 1970. Bacterial transformation, with special reference to recombination

J. BACTERIOL.

process. Annu. Rev. Genet. 4:193-224. 17. Michalka, J., and S. H. Goodgal. 1969. Genetic and physical map of the chromosome of Haemophilus influenzae. J. Mol. Biol. 45:407-421. 18. Notani, N. K., F. R. Frankel, and S. H. Goodgal. 1965. On the association of donor and recipient DNA's during transformation in Haemophilus influenzae. Proc. Symp. on the Mutational Process, Prague, p. 151-157. 19. Notani, N. K., and S. H. Goodgal. 1965. Decrease in integration of transforming DNA of Haemophilus influenzae following ultraviolet irradiation. J. Mol. Biol.. 13:611-613. 20. Postel, E. H., and S. H. Goodgal. 1972. Competence mutants. II. Physical and biological fate of donor transforming DNA. J. Bacteriol. 109:292-297. 21. Postel, E. H., and S. H. Goodgal. 1972. Competence mutants. m. Responses to radiation. J. Bacteriol. 109: 298-306. 22. Setlow, J. K., M. E. Boling, and K. L. Beattie. 1970. Repair of DNA in Haemophilus influenzae. Ill. Excision and recombination defects and the site of repair of ultraviolet-irradiated transforming DNA, p. 555-569. In Genetic concepts and neoplasia. 23rd Annual Symposium on Fundamental Cancer Research, Houston, Texas. Williams & Wilkins Co., Baltimore. 23. Steinhart, W. L., and R. M. Herriott. 1968. Genetic integration in the heterospecific transformation of Haemophilus influenzae cells by Haemophilus parainfluenzae deoxyribonucleic acid. J. Bacteriol. 96:17251731. 24. Stuy, J. H. 1965. Fate of transforming DNA in the Haemophilus influenzae transformation system. J. Mol. Biol. 13:554-570. 25. Voll, M. J., and S. H. Goodgal. 1965. Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae. J. Bacteriol. 90:873-883.