GENETIC STUDIES 95 AN ESCHERICHIA VCGLI WIFE-f A ISUPPRESSED R FACTOR AND PHYSICAL STUDIES OF A NUQLEGPRQ‘FEIN FMM AME: CQLI CONTAINING THE UNSUPPRESSED R FAC'IOII TI’IGSIS Io» I’Im Devon of pk. D. MICHIGAN STATE UNIVERSITY Fred Truby Hickson 1965 THESIS (J . f). LIBRARY Michigan State University This is to certify that the thesis entitled GENETIC STUDIES OF AN ESCHERICHIA COLI WITH A SUPPRESSED R FACTOR AND PHYSICAL STUDIES OF A NUCLEOPROTEIN FROM AN E. COLI CONTAINING THE UNSUPPRESSED R FACTOR presented by Fred Truby Hickson has been accepted towards fulfillment of the requirements for Ph.D ' degree in Microbiology and PUBIlC Health 1' ,- ' . z ' C“ , . / «div, Lax L a» /' t L L Major professor I" h ‘I ', ' [/16 /'K I 1 Date Willi; wt 5/ if: : I9 > T 0-169 ABSTRACT GENETIC STUDIES OF AN ESCHERICHIA COLI WITH A SUPPRESSED R FACTOR AND PHYSICAL STUDIES OF A NUCLEOPROTEIN FROM AN E. COLI CONTAINING THE UNSUPPRESSED R FACTOR by Fred Truby Hickson The purpose of this study was to try to learn what are the effects of a suppressor mutation upon the drug resistance factor (R) when it is in an autonomous cytOplasmic condition. Escherichia coli B-3 thy— (Rf) possessing re— sistance to chloramphenicol, streptomycin, and sulfathiazole was used for this study. The suppressor mutation was induced with nitrous acid. Accompanying the suppression of the phenotypic ex- pression of streptomycin and chloramphenicol resistance was the acquisition of a requirement for tetrahydrofolic acid or one of its derivatives and the loss of the capacity to pro— duce a colicin—like substance. The suppressor mutation did not affect the transfer— ability of the R factor to the recipient strain, E. 99;; Kr12 arg-T6r. The marker responsible for the suppressor mutation was not transferred in a mixed culture. Therefore, it is most likely a chromosomal mutation. Fred Truby Hickson Attempts were made to modify the Cairns (l963)*pro- cedure for the isolation of DNA in hope of obtaining DNA in its most native state. Double stranded DNA was isolated but it was bound to protein and considerably degraded according to its sedimentation band in a sucrose density-gradient. Therefore, it was not possible to study the species of DNA in the R—containing strain and the suppressor mutant derived from it. *J. Cairns, 1963. The chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. g§;43-45. GENETIC STUDIES OF AN ESCHERICHIA COLI WITH A SUPPRESSED R FACTOR AND PHYSICAL STUDIES OF A NUCLEOPROTEIN FROM AN E. 99;; 'CONTAINING THE UNSUPPRESSED R FACTOR BY Fred Truby Hickson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1965 ACKNOWLEDGMENTS I wish to express my deepest appreciation to Dr. Delbert E. Schoenhard for his encouragement, guidance, and extreme patience during the course of this study. I should also like to acknowledge the constructive suggestions given me by Dr. J. A. Boezi and Dr. J. L. Fairley. A special note of thanks is due Mr. I. L. Dahljelm and Dr. J. J. Stockton for their valuable assistance and advice. The cultures used in this study were supplied by Dr. H. S. Ginoza, National Aeronautics and Space Admini— stration, Ames Research Center, Moffett Field, California, to whom I would like to express my appreciation. ii This thesis is dedicated to my wife and children who gave me inspiration when needed iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 4 Transfer of multiple drug resistance by conjugation 4 Concept of the R factor . . 6 Concept of the episomal nature of the R factor 8 Spontaneous segregation of the R factor 9 Evidence of chromosomal attachment of the R factor . . . . . .. . . . . . 10 Evidence for suppression of Cm marker on R factor . . . . . . . . . . . . . . . . . . 11 Other suppressor mutations . . . . . . . . . . . . ll Folic acid mutants . . . . . . . . . . 13 General properties of a nucleoprotein . . . . . . l4 Classification of nucleoproteins . . . . . . . . . 15 General properties of bacterial DNA . . . . . . . 1? Isolation of bacterial DNA . . . . . . . . l8 Characteristics of a good deoxyribonucleic acid preparation . . . . . . . . . . . . . . . . 20 MATERIAL AND METHODS Cultures . . . . . . . . . . . . . . . . . . . . . 21 Media . . . . . . . . . . . . . . . . . . . . . . 21 Drugs . . . . . . . . . . . . . . . . . . . . . . 22 Mating procedure . . . . . . . . . . 22 Mutational procedure for selection of suppressor mutant . . . . . . . 23 Reselection of possible suppressed mutants . . . . 25 Growth requirement of a suppressor mutant . . . . 26 Curing of E. coli 3-3 thy‘ (R+) . . . . . . . . . 27 Salmon sperm DNA solution . . . . . . . . . 28 DNA isolation from‘g. coli B-3 thy (R+) cells using a modified Marmur procedure . . . . 28 Preparation of lysate by a modification of Cairns' s procedure . . . . . . . . . . 29 Deproteinization of lysate by phenol . . . . . . . 31 Chemical analysis of lysate . . . . . . . . . . . 32 iv Page Ultraviolet absorption spectrum of lysate . . . . 32 Denaturation of nucleoprotein . . . . . . . . 33 Preparation of histone from nucleic aCid . . . . . 33 Sedimentation of nucleoprotein . . . . . 33 Zone sedimentation in sucrose density- gradient . . 33 RESULTS Conditions of conjugation of E. coli B- 3 thy (R+) with E. coli K—12 arg ref“ . . . . 35 Nitrous acid-induced mutations of E. coli B- 3 thy (R+ ) . . . . . . . . . . . . . . . . . . . 39 Loss of mutants . . . . . . . . . 41 Reselection for mutants from refrigerated cultures . . . . . . . . . . . . . 44 Characteristics of haCk mutants . . . . . . . . . 45 Characteristics of a randomly chosen suppressor mutant . . . . . . . . . . . . 47 Test for growth requirement of E. coli B- 3 thy SuR (R+) . . . . . . . . . 47 Crosses of E coli+ B— 3 thy SuR - (R+) and E. coli B—3 thy (R+ ) with E coli K—12 arg T6r under almost identical conditions . . . 53 E. coli B— 3 thy (R+ ) cured of the R+ factor . . . 53 Deoxyribonucleic acid extracted from cells . . . . 56 Ultraviolet absorption spectrum of lysate . . . . 58 Composition of the lysate . . . . . . 61 Sodium hydroxide degradation of the lysate . . . . 61 Attempt to separate the protein from the DNA . . . . . . . . . . . 64. Attempt to remove nucleoprotein by centrifugation . . . . . . . . . 67 Sucrose density-gradient centrifugation . . . . . 67 Phenol treatment of the lysate . . . . . . . . . . 69 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 79 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 89 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 91 Table 10° 11. LIST OF TABLES Response to T6 phage Growth requirements Response to drugs Drug resistance of E. coli K-12 arg-T6r after conjugation with E. coli B-3 thy- (R+) Cross of E. coli B-3 thy-[SuRf-potential] (R+) mutant with E. coli KrlZ arg-T6r Resistance oqu. coli B—3 thy-[SuR--potential] (R+) mutants Growth requirement and fermentation patterns of the donor, the recipient, and a sup- pressor mutant of E. coli Transfer of resistance factor from one of the back mutants of E. coli B-3 thy-Su (R+) to E. coli K-12 arg-T6r R Substances which support the growth of E. coli B-3 thy-SuRI (R+) Growth requirements forE° col; B—3 - - + thy SuR (R ) Crosses of E. coli B—3 thy— (R+) and .E. coli B-3 thy_ SuR- (R+) with E. coli K—12 arg_T6r under similar conditions vi Page 36 37 38 4O 42 43 46 48 50 52 54 Table 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. 22. 23. 24. Cross of an E. Loli K-12 arg T6r (R+) with a cureduE. cLli B— 3 thy . . . . . Percentage of deoxyribonucleic acid ex- tracted from cells Deoxyribonucleic acid content of lysate calculated by O.D. conversion factor Ratios for absorption of DNAs at 260: 230:280 mp . . . . . . . . Substances present in the lysate compared to the amount of the same substances present in DNA isolated by a modified Marmur procedure and to those present in Salmon sperm DNA Calculation of the amount of degradation of the lysate with sodium hydroxide Assay by ultraviolet absorption of the ethanol precipitates of the lysate Assay by ultraviolet absorption of 0.7 ml fractions obtained after centrifugation at 114,743 x G for 2 hours Ratios for absorption at 230, 260, and 280 my of material from tubes indicated by arrows in Figures 3'and 4 Comparison of ratios for absorption an: 230, 260, and 280 mp of phenol—treated lysate with other DNAs . . . . . . Substances present in the phenol~treated lysate as compared to those present in the lysate and in the DNA isolated by the modified Marmur procedure . . . . Ratios for absorption at 230, 260, and 280 my of material from tubes indicated by arrows in Figure 6 Molecular weight of smallest gene as compared to molecular weight of nucleoprotein vii Page 57 59 59 62 63 65 66 68 72 74 76 78 87 Figure LIST OF FIGURES Circular models of the R factor Ultraviolet absorption spectrum of a 1:6 dilution of the lysate in 0 15 M NaCl plus 0. 010 M trisodium citrate plus 0. 010 M KCN at pH 7 0 . . . . Absorption at 260 mp of 0.7 m1 samples collected from a 5-30% sucrose density— gradient after the sedimentation of the lysate Absorption at 260 mp of 0.7 m1 samples collected from a 5—30% sucrose density- gradient after the sedimentation of the DNA extracted by a modified Marmur procedure . . . . . . . . . Ultraviolet absorption spectrum of a 1:2 dilution of the phenolctreated lysate in 0.15 M NaCl plus 0.010 M trisodium citrate plus 0.010 M KCN at pH 7.0 Absorption at 260 mp of 0.7 m1 samples collected from a 5-30% sucrose density- gradient after the sedimentation of the phenol-treated lysate viii Page 60 7O 71 73 77 INTRODUCTION Several Japanese researchers have found that drug resistance can be transferred from one organism to another by cell-to-cell contact without the accompaniment of any other markers (Watanabe, 1963). They have reported that the drug resistance markers are transferred together, replicate faster than the host chromosome, and can be eliminated from the organism without affecting the survival of the organism in any media except those containing drugs. It seems ap- parent that they are dealing with a factor that exists as a cytoplasmic element. They have also reported results in which it seems that the drug resistant markers become inte— grated with the host. Genetic elements which alternate be- tween integration upon the chromosome and autonomous repli? cation in the cytoplasm are called episomes. The drug resistance referred to here fits the category of an episome and is called the resistance factor (R). When a mutation was induced 1n the chloramphenicol marker of the drug resistance factor of a thymine—requiring strain of Escherichia coli by the author of this paper, a de- crease in the concentration of chloramphenicol to which this organism was now resistant was observed. However, when this organism was used as a donor and an arginine-requiring strain of E._EQEE K-12 was used as the recipient, it was observed that the concentration of chloramphenicol to which the re- cipient became resistant was the same as if the non-mutated thymine—requiring strain of E. Egli had been used as the donor. Thus, it appeared that there might be a suppressor mutation on the chromosome of the bacteria which would in- hibit the expression of an episomal element~—the drug re- sistance factor. This same effect had been noted by Watanabe and Fukasawa (1961c) when the chloramphenicol locus from a strain containing the drug resistance factor was trans- duced to E. Egll W_ll77. Thus, it was decided to attempt to mutate the thymine- requiring stra1n of E. 29;; that contained the drug resistant factor to a suppressed condition which would affect more than just the chloramphenicol locus. Once this had been ac- complished, it would be necessary to determine if any other markers were involved in the suppressor mutation and to see if the suppressor could be transferred into a recipient cell along with the drug resistance factor. Mutants wh1ch pos- sessed suppressor mutations that affected the expression of the R factor were isolated and were shown to be independent of the cytoplasmic R factor. From this point, it was decided to attempt to sepa- rate the episomal element from the chromosomal element using a mild extraction procedure and sucrose density-gradient centrifugation. It was necessary to use a technique that would not cause shearing of the chromosome upon lysis of the cells and thus not produce chromosomal fragments which would be similar 1n size to episomal particles. In view of this requirement, a modification of the procedure used by Cairns (1963b) was tried. Lfthis procedure was successful, it*was planned to determine by the sucrose density—gradient tech- nique whether there was any difference in the molecular weights of the episomal elements of the suppressed strain and the parent strain as well as to see if a cured strain still possessed material which could be considered episomal. LITERATURE REVIEW Transfer of Multiple Drug Resistance by Conjugation In 1959, several Japanese investigators showed that drug resistance to chloramphenicol (Cm), streptomycin (Str), tetracycline (Tc), and sulfathiazole (Sul) could be trans- ferred from resistant Shiqella dysenteriae to Escherichia coli and vice versa by merely mixing the two cultures to- gether. Since all attempts to transfer the resistance with cell-free filtrates of the resistant cells failed, they con- cluded that cell-to-cell contact was essential for the trans— fer. It was also noted by these investigators that neither biochemical nor serological markers were transferred during the transfer of the drug resistant markers (Watanabe, 1963). Watanabe and Fukasawa (1961a) found that these drug resistant markers of a few cells mixed with quite a few re- cipient cells would soon appear amcng all of the recipient cells before cell division had had an opportunity to occur. They thus concluded that the factor involved in the transfer of drug resistance must replicate autonomously 1n the cyto— plasm of the donor cells. Some other Japanese investigators had isolated multiple drug resistant mutants by exposing sensitive E. 39;; strains to the individual drugs, one after the other (Watanabe, 1963). They then attempted, without success, to transfer this resistance on to a sensitive recipient by mixed cultivation. Watanabe and Fukasawa (1961a) had found also that the frequency of transfer of the drug resistant markers differed considerably from donor to donor and also from recipient to recipient. They found that the F- strains of E, 39;; K-12 were the best recipients. A frequency of transfer per hour per donor cell ranged from 10-2 to 10-7, depending on the donor strain employed. Some of these donor strains were also found to produce colicins and phages, which reduced the frequency of transfer by killing recipient cells. They noted that in most cases the drug resistant factors received by the recipient were expressed phenotypically before the first cell division. The only exception was in the Str resistance marker which in some recipient cells required a cell division before being expressed. Although Watanabe and Fukasawa (1961a) had found that the levels of resistance to the drugs varies consider- ably among strains, there was no evidence that the resistance factors were modified by the host cells during their transfer. They also showed that the transfer of multiple drug resistance could be interrupted by treatment of the cell mixture with a blendor which added proof that the transfer was brought about by conjugation. Since no gradient of transfer of the drug resistant markers could be found even when the conju— gating mixture was treated at an early stage with the blendor, they concluded that the drug resistant markers must be closely linked and transferred very rapidly. These find- ings along with the others led Watanabe and Fukasawa (1961c) to suggest that the resistance factors replicate in the cyto- plasm independent of the host chromosome and are transferred by conjugation. Concept of the R Factor Watanabe and Fukasawa (1961c) reported that the order of entry of the drug resistant markers seemed to be either Cm-Str-Sul-Tc or Sul-Str—Cm-Tc. Since this order of drug resistant markers was not found on the chromosome of any strain oqu. coli, Shiqella dysenteriae, or Salmonella typhimurium, they assumed that the resistance factors must have originated from thechromosome of some other bacteria. In the model proposed by Watanabe (1963) and shown in Figure l, the R factor is used to denote the drug resistant markers along with the RTF (resistance transfer factor) which is the marker that is necessary to bring about the transfer of the other markers. Thus, this RTF marker must occupy a particu- lar position on the R factor and is possibly the point of integration of this particle with the host genome. Sul Str Cm Tc RTF or Cm Str Sul c RTF Figure 1. Circular models of the R factor (Watanabe, 1963). This type of circular model was also proposed for other episomal particles-—the F factor, lambda phage, the colicinogenic factor, and the F' factor—-by Campbell in 1962. However, all data thus far available on the R factors could be just as well understood using a linear model (Watanabe, 1963). Concept of the Episomal Nature of the R Factor The term episome was first used by Thompson (1931) in his definition of a gene in Drosophila as consisting of a main particle ("protosome") firmly anchored in the chromosome with varying numbers of one or more kinds of other particles attached ("episomes"). It was recently returned to active use by Jacob and WOllman (1958) to explain the behavior of temperate phage, fertility factors, and colicinogenic factors. The current operational definition of an episome is that it is an independent genetic structure that is additional to the normal chromosome of the cell which it inhabits, is transmissible by infection, and is propagated in alternative states--either autonomously in the cytoplasm or in associ- ation with the host chromosome (Hayes, 1964). In the autono— mous state, the episome would replicate independent of the host chromosome and usually at a faster rate. In the inte- grated state, the autonomous replication of the same episome would be inhibited and would replicate at the same pace as the chromosome (Jacob, Schaeffer3 and Wollman, 1960). The R factor may be included in the category of an episome by the following characteristics. First, it has the ability to pass from cell—to-cell very rapidly during conju— gation without the accompaniment of any chromosomal markers. Second, the transfer of the R factor can be interrupted by treatment with the Waring Blendor without segregation of any of the drug resistant markers. Third, the very rapid and indefinite transfer of the R factor from sensitive strain to sensitive strain without host modification of the factor indicates that the resistance factor replicates faster than the host genome. Fourth, the recombinants obtained from an Hfr (R+) x F— cross that had received the segment of the host chromosome between thiamine and mannitol were found to have received the R factor at a low frequency. Finally, treatment with acridine dyes brought about the elimination of the R factor from some organisms in the same manner as it eliminates other episomes (Watanabe, 1963). Spontaneous Segregation of the R Factor The spontaneous loss of part or 211 of the R factor from an organism has been noted by many investigators (Watanabe, 1963). Watanabe and Fukasawa (1961b) found that when cells with the multiple drug resistance factor were incubated in broth containing penicillin and Cm, they were able to isolate spontaneous segregants. Some of these 10 spontaneous segregants were found to have lost all of their drug resistant markers. Thus, the authors suggested that spontaneous segregation of the R factor might have been brought about by genetic exchange between the RTF-carried resistance factor and the host genome. It was assumed that the genetic exchange took place when the R factor was in the integrated state, in which condition autonomous replication of the R factor in the cytoplasm is probably inhibited. Evidence of Chromosomal Attach- ment of the R Factor Japanese investigators studied the transfer of host chromosomal markers and the R factor by Hfr strains contain— ing an R factor. When they selected recombinants resulting from chromosomal transfer by conjugation and examined them for drug resistance, only those recombinants which received the segment of the host chromosome between the B1 and mannitol loci were resistant. Thus, they concluded that the R factor was integrated at a specific site between B1 and mannitol and was transferred to the recipient cell along with the host chromosome. There have also been other reports which indicated that another point of attachment for the R factor may be near the site of the chromosomal attachment of the F factor (Watanabe, 1963). 11 Evidence for Suppression of Cm Marker on R Factor Watanabe and Fukasawa (1961c) noted that when Plkc phage was propagated upon an E. 32;; possessing an R factor with Sul, Cm, Str, and Tc markers and was used to transduce .§-.22ll W91177, transductants did not appear on Cm plates and most of the transductants selected on Tc plates were found to have only slight resistance to Cm. Furthermore, W—ll77 cells that received the Cm resistance factor by conju- gation also showed only slight resistance to Cm. However, these cells were found to be able to transfer the Cm marker to E. 22;; CSH—2 which then became very resistant to Cm. The investigators, however, did not investigate this apparent suppression any further. Other Suppressor Mutations A suppressor mutation has been defined as a mutationally altered gene that reverses the effect of a pri— mary mutation in the same or separate gene (Hayes, 1964). The action of a suppressor mutation may involve a number of loci having different or even, apparently, unrelated functions, or may be restricted to those alleles of a single locus, or even be allele specific (Hayes, 1964). Some ex- amples of the markers for which a suppressor has been found are: the A protein of tryptophane synthetase in E. 22;; (Yanofsky and Crawford, 1959), the ambivalent mutants of the 12 rII region of T4 phage (Benzer and Champe, 1961), alkaline phosphatase 0f.§-.£2£l (Garen and Siddiqi, 1962), and in Salmonella typhimurium the adenine—thiamine locus (Yura, 1956), the leucine locus (Smith—Keary, 1960), and the cysteine locus (Howarth, 1958). Hashimoto (1960) also found that there were 16 different suppressor alleles in a closely linked locus in E. 29;; that would suppress all of the clustered mutations to either Str dependence or Str resistance. In the suppressor mutations of the A protein of tryptophane synthetase, Yanofsky and Crawford (1959) showed that there was the formation of a protein or the re— establishment of enzymatic activity which returned the mutant to wild-type activity. The same authors also showed that the suppressor gene could alter the specific amino acid in- corporation into the A protein and hypothesized thatthis was due to a difference in the transfer RNA (Crawford and Yanofsky, 1959). Thus, it appeared that a suppressor mutation may lead to a change in the transfer RNA molecules so that they accept the "wrong" amino acid. Consequently, trans- lation of the m-RNA will produce proteins or enzymes pheno— typically similar to wild-type. On the other hand, Crick g; §l° (1961) showed that reversionsxmfacridine-induced mutations of the rII mutants of phage T4 were always by suppressor mutations at a different but closely linked site. Thus, the authors 13 postulated that suppressor mutations worked by the addition of a base where one had been deleted or, vice versa, so that the proper wild-type number of bases was restored. Folic Acid Mutants Folic acid and its derivatives were found to act as essential growth factors for a few microorganisms (Guirard and Snell, 1962). The requirement for folic acid and its derivatives by microorganisms was shown by Stokstad (1941) and Stokes (1944) to be eliminated by the addition of thymine and purine bases to minimal media. These findings indicated that folic acid functioned in the formation of these com- pounds. Additional studies by Rabinowitz and Himes (1960) showed that tetrahydrofolic acid derivatives functioned as the carrier of l-carbon units that become involved in the synthesis and degradation of purine bases, thymine, serine, and methionine. The biosynthesis of folic acid was found by Lascelles and Woods (1952) to procede through p—aminobenzoic acid and that the reaction which utilized this metabolite was in- hibited by sulfonamides. Leucovorin, N5 formyl tetrahydrofolic acid, was used to overcome the folic acid requirement in several micro- organiSms (Bond _E él-: 1949). Leucovorin is a synthetic compound that is a mixture of diasterioisomers with one-half the biological activity of the citrovorum factor or N5 formyl l4 tetrahydrofolic acid (Guirard and Snell, 1962). The pathway of incorporation of leucovorin into the tetrahydrofolic acid pathway is outlined by Oginsky and Umbreit (1959) as: Folic acid 1 Tetrahydrofolic acid 5’10 methylenyl tetrahydrofolic acid 1 formyl tetrahydrofolic acid N N5,10 / Leucovorin Purine synthesis General Properties of a Nucleoprotein Deoxypentose nucleic acids have often been isolated in the form of a nucleoprotein. A deoxynucleoprotein is de- fined as a conjugated protein in which the union of the deoxyribonucleic acid, which functions as the prosthetic group, and the protein is mediated by electrostatic at— traction or by secondary valence forces. A decision, how— ever, must be made in every case as to whether what one has isolated really pre-existed in the cell or whether the combination between the protein and the DNA is simulating a definite compound (Chargaff, 1955). 15 In the absence of a proper biological test procedure, there is really no way of determining what is the native state of a nucleoprotein. The nucleic acid content, 35—60% of the nucleoprotein's dry weight, seems to be a function of the source of the nucleoprotein and the means of its sepa— ration. The absorption spectrum of nucleoproteins in the ultraviolet region is, in general, identical to that of the nucleic acid itself with a maximum around 260 my. However, it does differ from the nucleic acid spectrum in that it rises sharply again at a point below 245 mp and keeps rising. This rise is caused by the presence of the protein whose ab- sorption is no longer negligible in this range when compared to the nucleic acid (Mirsky and Pollister, 1946). Several physical criteria—-viscosity, ultracentrifu- gation pattern, electrophoretic behavior, light scattering, and ultraviolet absorption-—have been used to determine the integrity of a nucleoprotein preparation. From these tests, it is possible to learn how degraded a given specimen is but not how intact it is (Chargaff, 1955). Classification of Nucleoproteins ,iChargaff, 1955) The nucleoprotamines--These have been isolated from the spermatozoa of many genera of fish. Solutions of high ionic strength are used to isolate them. In this condition, there is a marked separation of the nucleic acid from the 16 protein. Here, advantage is taken of the fact that the high salt concentration tends to suppress DNase activity. The nucleoprotamines are soluble in 1 M sodium chloride but in— soluble at the physiological saline concentration. The nucleohistones——These have been isolated from bird erythrocytes, the thymus gland, and from pea embryos. In order to avoid conditions that cause dissociation of the nucleic acid and the protein, low ionic strength solutions are used to isolate them. There exists, however, the danger of enzymatic degradation brought about by the release of the nucleases. Thus, the nucleohistones must be isolated in the presence of arsenate, citrate, or ethylenediaminetetra ace- tate in order to suppress this nuclease activity. These nucleohistones also have a minimal solubility at the physio— logical concentration of sodium chloride but an increasing solubility as the ionic strength is either raised or lowered from this point. The nucleoproteins--This type of a nucleoprotein has been isolated from avian tubercle bacillus. Here, the deoxypentose is bound to a protein which lacks the basic properties of protamines or histones. These nucleoproteins are soluble in physiological saline and by this property, they differ from the nucleoprotamines and nucleohistones. 17 General Properties of Bacterial DNA The DNA of bacterial cells represents about 3-4% of the dry weight, around 10-14 g/cell in.§-.EQli- This repre— sents about 1000 molecules of DNA if its molecular weight is 8 x 106 (Luria, 1960). According to these figures, if the bacterial genome were a single unit, it would have a molecu- lar weight of 8 x 109. Recently, Cairns (1962) lysed bacterial cells which had labelled DNA and the total counts of the grains per chromosome of the autoradiographs was con- verted into a molecular weight which for a single genome came out to be around 2.8 x 109. By phosphorus decay, Fuerst and Stent (1956) calculated the molecular weight of DNA of an E. gal; cell as being 4 x 109. The reason for obtaining uniformly low molecular weight fragments (8-30 x 107) from E. EQEE, prior to the work of Cairns, was thought due to the breakage of possible pre-existing breakage points, such as protein links, which are built into the genome at uniformly spaced points (Cairns, 1962). The possibility of weak points in the DNA molecule has been demonstrated by Hershey and Burgi (1960) in experi- ments in which shearing forces were applied to a DNA prepa— ration. They found that half molecules can be produced re- peatedly until some size is reached that will survive the shearing force being applied. l8 Isolation of Bacterial DNA Marmur's method (Marmur, l961)-—Cells are disrupted in the presence of a sodium lauryl sulfate solution at a high pH (8.0). These cause a suppression of enzyme activity and denaturation of some of the proteins. The denatured protein and cell debris are removed by centrifugation. Further denaturation of the proteins has been carried out by phenol instead of the chloroform—isoamyl alcohol procedure utilized by Marmur (Saito and Muira, 1963). Ribonucleic acid is then removed by DNase—free RNase (Bonhoeffer and Gierer, 1963) and by selective precipitation of the DNA with isopropanol. The degradation of DNA by DNase in the presence of divalent metal ion contamination is suppressed throughout the procedure by the presence of a chelating agent and by the action of sodium lauryl sulfate. Cairn's method (Cairns, 1962; l963a; and l963b)—- Degradation of DNA prepared by Marmur's method could take place at (1) the time of lysis, because the cells probably break open with a sudden burst, (2) during the deproteini- zation step when the cell extract is stirred with phenol, and (3) the winding of the DNA from the isopropanol solution by stirring with a glass rod. Thus, a milder treatment to lyse the cells was utilized by Cairns in order to minimize the shearing forces applied to the DNA. 19 Cells were treated with lysozyme instead of dupanol in order to leave the DNA complexed with the basic proteins and protamines and thus less likely to breakage by turbulence. While the cells were being treated with lysozyme, they were suspended in 1.5 M sucrose which was then gradually lowered to the point of extinction. It was also carried out in the Eresence of ethylenediaminetetra acetate and a high pH (8.0) in order to chelate divalent ions and thus suppress DNase activity. After lysis was completed, the salt concentration was raised to 4 M in order to remove and precipitate the non— specifically adsorbed proteins from the nucleic acid. Finally, the lysate was treated with RNase in order to eliminate most of the RNA. Berns and Thomas (1965) used the mild isolation pro- cedure of Cairns; however, they added a deproteinization step. The deproteinization was accomplished by twice ex- tracting the lysate with equal volumes of water-saturated phenol in a screw-capped test tube. During each extraction, the material in the test tube was revolved on a drum at 60 rev/min for 60 min at 4 C. After the second extraction, the aqueous layer was gently extracted once with an equal volume of non-anhydrous ether and then dialyzed overnight against 0.15 M sodium chloride plus 0.015 M sodium citrate. 2O Characteristics of a Good Deoxyribonucleic Acid Preparation Chargaff (1955) describes the characteristics of a good DNA preparation as follows: (1) absence of proteins, polysaccharides, lipids etc., (2) absence of pentose nucleic acids, (3) a maximum absorption between 257 and 261 mp of a solution at pH 7.0, (4) a fibrous character, a high viscosity, and the presence of streaming birifringence, (5) monodisper- sity in the ultracentrifuge, and (6) electrophoretic homogeneity. MATERIALS AND METHODS 2E1.-t_u_rs§ The bacterial strains and their genetic character— istics were Escherichia coli B—3 thy- (R+) which possessed a resistance to 50 pg/ml chloramphenicol, 25/pg/m1 sulfa- thiazole and §°.£2l£ K-12 F— arg- which was mutated spon— taneously to T6r (referred to in this paper as E. 39;; K—12 arg_T6r). A T6 phage stock at 2.1 x 1012, prepared by the procedure described in Adams (1959), was used to counter- select the donor. Media Penassay broth (PA), Purple Broth Base, and nutrient broth (NB), all dehydrated products of Difco, were used. Purple Broth Base was supplemented with 1% carbohydrate and nutrient broth was supplemented with 0.05% NaCl and 0.01% dextrose. The synthetic medium described by Davis and Mingioli (1950) enriched with 20'pg/ml of the required nutrient was utilized throughout this work. Solid or semi- solid media were prepared by the addition of 1.5% or 0.6% agar, respectively, to the above broths. Supplements of either nutrients or agar were added on a weight per volume basis. 21 22 Drugs Stock solutions of chloramphenicol, penicillin and streptomycin were prepared as follows: (1) 1000 mg of chloramphenicol (Cm) (Parke, Davis and Company) were first dissolved in 10 ml of propylene glycol and then brought up to 500 m1 using distilled water, (2) 200 mg of dihydro— streptomycin sulfate (Str) (E. R. Squibb and Sons) were dis- solved in 100 m1 of distilled water, and (3) buffered peni— cillin G potassium (E. R. Squibb and Sons) was prepared by adding 4 m1 of distilled water to a vial containing 200,000 units of penicillin. Sulfathiazole (Merke and Company) was weighed out just prior to its addition to the media and was added to the media just after it had been removed from the autoclave. Mating Procedure A 0.1 m1 sample of an overnight culture of the re- cipient and the donor were each inoculated into separate test tubes containing 10 ml of PA (NB was used with suppressor mutants) and incubated without aeration at 37 C until there were 2—5 x 108 cells/m1. Samples were then withdrawn from the test tube, diluted in physiological saline, and plated to obtain a viable count as well as a count of the drug re- sistant cells. At the same time, 1 ml of both the donor and the recipient were placed in a 250 m1 Erlenmeyer flask that 23 contained 8 m1 of PA broth. This flask was then incubated without aeration in a 37 C water bath for 30-60 minutes. At the end of the conjugation period, T6 phage were added to the flask in a concentration of 100 phage per donor cell and the flask was incubated an additional 30 minutes at 37 C. Upon the completion of the counterselection, the cells were centrifuged, washed 2 times with physiological saline, and resuspended in 10 ml of Davis synthetic media plus the growth requirement for the recipient. This culture was then incubated for another hour without aeration at 37 C and then centrifuged, washed, diluted in physiological saline, and plated on selective media containing each of the drugs as well as on non-selective media. Inoculated nutrient agar plates were incubated for 24 hours before being observed; whereas, minimal agar plates were incubated for 48 hours (72 hours when using Davis synthetic medium plus thymine and leucovorin). Mutational Procedure for Selection of Suppressor Mutant One ml of an overnight culture of E. EQEE B—3 thy-(R+) was inoculated into 9 m1 of NB whose pH had previously been adjusted to a pH of 4.5. To this culture was then added 1 m1 of 2 M NaNO and the tube was incubated without aeration for 2 90 minutes at 37 C. The culture was then filtered through a Millipore filter (0.45‘p pore size) followed by four-10 ml aliquots of 0.038 M phosphate buffer at pH 7.0. 24 The filter was then placed in a sterile 150 mm beaker that contained 10 ml of Davis synthetic medium plus 20 pg/ml of thymine along with 25 pg/ml of Str. The beaker was then agitated on a Vortex Jr. Mixer for one minute and the liquid was pipetted into a sterile test tube. After this test tube had been incubated without aeration for 2 hours at 37 C, 0.4 m1 of penicillin (50,000 units/m1)*wereadded and incubation was continued for an additional 3 hours (Gorini and Kaufman, 1960). Again, the culture was filtered through a Millipore filter (0.45 p pore size) followed by two—10 m1 aliquots of distilled water. The filter was placed in a sterile 150 m1 beaker containing 5 ml of physiological saline and agitated on a Vortex Jr. Mixer for one minute. The liquid was then transferred to a sterile test tube and 0.1 ml was plated on several nutrient agar plates. After incubating the nutrient agar plates for 24 hours at 37 C, the colonies were replicated (Lederberg and Lederberg, 1952) onto nutrient agar plates containing 25 ‘pg/ml of Str. These plates, after incubation for 24 hours at 37 C, were compared to the nutrient agar plates. Any colonies that did not develop on the Str plates were picked from the nutrient agar plates and spread in small patches on— to both nutrient agar plates and nutrient agar plus Str plates. 25 Absence of observable growth on a streptomycin agar plate in an area previously inoculated with cells from an isolated colony and observable growth on a nutrient agar plate previously inoculated with cells from the same isolated colony were taken as evidence of drug sensitivity of the cells of the selected colony. Cells were transferred from the growth of the suspected drug sensitive mutants to 3 m1 of PA broth. After these cultures had grown to a concentration of 1 x 108, they were tested for their fermentation pattern and were crossed with §-.22ll K-12 arg-T6r. Reselection of Possible Suppressed Mutants A loopful of the possible suppressor mutants was streaked on nutrient agar plates and incubated for 24 hours at 37 C. Since the mutant colonies were smaller than were the back mutant colonies, one of the smaller colonies was picked and inoculated into 1 m1 of physiological saline. One-tenth m1 of the saline suspension was then inoculated in— to NB, PA, and Davis synthetic medium supplemented with 20 ‘pg/ml of thymine and 1.5% casamino acid (Difco). These cultures were then incubated without aeration at 37 C for 4 hours and then 0.05 mlvmnxaspread over a 1 sq cm area of nutrient agar plus 25 pg/ml Str plates. If the mutant did not grow on the Str plates, it was again tested for its fermentation pattern and crossed with_E. 29E; K-12 arg-T6r. 26 The broth cultures described above were then stored in the refrigerator for a period of a month and, periodically, samples were removed to determine the number of back mutants per ml. Nutrient broth was then utilized to propagate and store these mutants. Growth Requirements of a Suppressor Mutant Several plates of Davis synthetic medium plus 20 ‘pg/ml of thymine were covered with 2.5 m1 of soft agar inocu- lated with 0.1 m1 of a twice-washed overnight culture of the possible suppressor mutant 0f.§-.22ll B-3 thy_ (R+). These plates were allowed to dry for several hours. Crystals of four different amino acids and/or vitamins were then positioned equidistant from each other on the surface of the plates. These plates were then incubated for 72 hours at 37 C before being observed. After a block in the tetrahydrofolic acid pathway was indicated, Davis synthetic medium was supplemented with (1) varying concentrations, 0.2, 2.5, 10, and 20 pg/ml, of calcium leucovorin; or (2) 20.pg/m1 of DL-methionine, DL— serine, adenine, and guanine as a unit or individually. The inoculation procedure and incubation of the plates were the same as for the crystal test. 27 Curing of E. coli B-3 thy— (R+) Five m1 of Davis synthetic medium plus 20 pg/ml of thymine and 5 pg/ml of streptomycin were inoculated with 0.1 m1 of an overnight culture of.E._ggEE B-3 thy- (R+) and incu— bated for 2 hours. Four-tenths of a m1 of penicillin (50,000 units/m1) were added and incubation continued for an addition- al 3 hours without aeration. The culture was then filtered through a Millipore filter (0.45 p.pore size) followed by 2 washes with 10 ml aliquots of distilled water. The filter was then placed into a sterile 150 ml beaker containing 5 m1 of physiological saline and agitated by a Vortex Jr. Mixer for 1 minute. One-tenth of a m1 of the suspension was then plated on each of 10 nutrient agar plates using the spread plate technique. The plates were incubated at 37 C for 24 hours and then replicated onto nutrient agar plates containing 5-pg/m1 of Str. After these plates had been incubated for 24 hours at 37 C, they were compared to the nutrient agar plates. Any colonies which ap— peared on the nutrient agar plates but not on the Str plates were picked and grown in PA broth for 4 hours at 37 C. The PA cultures were then centrifuged, the cells washed 2 times with physiological saline, and resuspended in 5 ml of physiological saline. The saline suspensions were then streaked on Davis synthetic medium without supplemen— tation or supplemented with 20 pg/ml of thymine plus either S‘pg/ml of Cm or 50‘pg/m1 of Sul. If no growth developed on 28 these plates after 48 hours of incubation at 37 C, the organisms were spread on plates of Davis synthetic medium supplemented with 20 pg/ml of thymine to determine whether they were still of the original genotype. The "cured" E. coli B-3 thy- was then crossed with both E. coli K—12 arg-T6ras well as with E. coli Kr12 arg—T6r (R+). Salmon Sperm DNA Solution This was prepared by dissolving 10 mg of Salmon sperm DNA (Nutritional Biochemicals Corp.) in 90 m1 of dis— tilled water and then adding to it 10 m1 of a solution con- taining 1.5 M NaCl plus 0.15 M sodium citrate at pH 7.0. DNA Isolation from E. coli B-3 thyf (R+) Cells Usinqpa Marmur Procedure The procedure described by Marmur (1961) was utilized for the isolation of DNA from E. 29;; B—3 thy- (Rf) with two modifications. First, lysis of the cells was accomplished by adding 400 mg of sodium lauryl sulfate to 40 m1 of the cells suspended in 0.15 M NaCl plus 0.1 M Ethylenediaminetetra acetate at pH 8.0 and slowly stirring the mixture for 4 hours at 4 C on a magnetic stirrer. Second, the deproteinization steps were all done with phenol as described by Saito and Muira (1963) instead of the perchlorate—chloroform-isoamyl alcohol method used by Marmur. 29 Preparation of Lysate by a Modification of Cairns‘s Procedure Ten ml of an overnight culture 0f.§°.£2ll B—3 thy_ (R+) were used to inoculate 2 liters of nutrient broth supplemented with 20 pg/ml of Str. This culture was allowed to incubate at 37 C for 6 hours at which time the cell population had reached a density of 1-2 x 109/m1. The culture was then harvested by centrifugation and washed twice with a cold Saline-EDTA solution (0.1 M NaCl plus 0.1 M Ethylenediaminetetra acetate at pH 8.0). After collecting the last wash by centrifugation, the cells were resuspended in a total volume of 20 ml of Sucrose-EDTArKCN (1.5 M sucrose plus 0.01 M EDTA plus 0.01 M KCN at pH 8.0). The total contents were then placed in a cylindrical chamber with a 35 mm diameter and a 26 mm depth, faced on both sides with a VM Millipore filter (50 mp pore size). The enclosed bacteria were then dialyzed at 37 C against 200 pg/ml of lysozyme (Armour Pharmaceutical Co.) in 500 m1 of Sucrose-EDTA—KCN for 8 hours at 37 C. The contents in the dialysis chamber were then re— moved by a large bore pipette (made by breaking off the tip of a 10 m1 pipette) fitted with a propipette and placed into dialysis tubing with a 5/8th inch diameter. This tubing was then tied loosely at the tOp with string and was only filled approximately 1/3 full so that increases in the volume inside the tubing would not cause increased pressure. The tubing 30 was then placed back into the Sucrose-EDTAeKCN solution and the sucrose concentration was brought slowly down to 0.045 M by the removal of an aliquot of the dialyzing solution and adding back of an equal volume of Saline-EDTA—KCN solution (0.1 M NaCl plus 0.01 M EDTA plus 0.01 M KCN at pH 8.0)° This process took place at room temperature over a period of 10% hours and a drop in the sucrose concentration did not exceed more than 0.03 M at any exchange step. Once this process was completed, 10_pg/m1 of RNase, 5X crystallized (bovine pancreas), which had been previously treated by the procedure described by Bonhoeffer and Gierer (1963) to inactivate any contaminating DNase, was added to the material in the dialysis tubing and the temperature of the system was raised to 37 C for 2 hours. The material was then dialyzed against two changes of a Saline—EDTA-KCN solution at room temperature and then slowly (0.2 M incre- ments at a time) brought up to a NaCl concentration of 4 M by the addition of granular NaCl to the solution outside the dialysis bag. Once the NaCl concentration had reached 4 M and had been held at this concentration for 2 hours, the contents of the dialysis bag were removed by a large bore pipette fitted with a propipette and were placed into a 50 ml centrifuge tube. This tube was then centrifuged at 17,300 x G for 15 min in aa refrigerated Sorvall (4 C). The supernatant fluid was then removed, again with the large bore pipette 31 fitted with a propipette, and placed into a new dialysis tubing bag, tied loosely at the top with string, and placed back into the 4 M NaCl solution at room temperature. The NaCl concentration outside the dialysis bag was then slowly lowered to 0.25 M (by 0.2 M increments at a time) by the removal of the dialyzing solution and replacimgit with an equal volume of a Saline-EDTA—KCN solution. Upon reaching 0.25 M NaCl, the dialysis bag was dialyzed against a Saline- EDTA—KCN solution at pH 7.1 for 2 hours at room temperature. This was again repeated with a fresh Saline—EDTA—KCN solution. Finally, the lysate in the dialysis bag was removed with the pipette-propipette set—up and placed into the glass cylindrical chamber fitted on both sides with new VM Milli- pore filters (50 mp pore size). The chamber was then dialyzed against 2 changes at 4 hour intervals of a solution of Saline-Citrate—KCN (0.1 M NaC1 plus 0.01 M sodium citrate plus 0.01 M KCN at pH 7.1) at room temperature. At the end of this time, the lysate was removed by the pipette—propipette apparatus, placed into a sterile screw-capped test tube, and stored in the refrigerator at 4 C. Deproteinization of Lysate by Phenol The lysate was extracted twice with an equal volume of water-saturated redistilled phenol at 4 C. The screw- capped test tube that contained this mixture was rolled continuously at 60 rev/min (Frankel, 1963 and Berns and Thomas, 1965). The aqueous layer was then extracted two additional times with an equal volume of non-anhydrous ether for 10 minutes with continuous rolling at 60 rev/min at 4 C. Finally, the aqueous layer was dialyzed against two changes at 4 hour intervals of Saline-Citrate-KCN solution at 4 C. Chemical Analysis of Lysate The components of the lysate and the phenolized ly- sate were measured as follows: DNA was measured by the diphenylamine reaction (Burton, 1956), RNA was measured by the orcinol reaction (Schneider, 1956), and protein by both the Folin reagent (Lowry _E _E., 1951) and the micro-Biuret reagent (Zamenhof, 1957). Standards utilized in the measure— ments were respectively, Salmon sperm DNA (Nutritional Bio_ chemicals Corp.), sodium ribonucleate (Nutritional Biochemi- cals Corp.), and lysozyme (Armour Pharmaceutical Co.). The DNA content was also determined from the 260 mp O.D. reading by the formula set down by Ganesan and Lederberg (1964). Ultraviolet Absorption Spectrum of Lysate These were made on a Beckman DU using a 1 ml cuvette and blanked against Saline—Citrate-KCN solution. 33 Denaturation of Nucleoprotein The procedure described by Hotchkiss (1957) utilizing NaOH was used to denature the nucleoprotein. The Beckman DU was used to follow any change in O.D. brought about by the denaturation of the nucleOprotein. Separation of Histone from Nucleic Acid The procedure described by Crampton_§§_gl. (1954) was utilized in an attempt to separate the nucleic acid from the protein. Sedimentation of Nucleoprotein One ml of the lysate was placed in 3.5 m1 of Saline» Citrate-KCN solution and centrifuged at 114,743 x G for 2 hours in the Spinco model L ultracentrifuge using the SWL39 rotor at 10 C. Following the centrifugation, 0.7 m1 samples were removed from the top using a Pasteur pipette fitted with a rubber dropper bulb. These samples were then placed in 0.7 m1 of a Saline-Citrate-KCN solution and read on the Beckman DU at a wavelength of 260 mp (Petermann and Lamb, 1948). Zone Sedimentation in Sucrose Density-Gradient The procedure described by Berns and Thomas (1965) was employed with the modification that all centrifugations 34 were made using SW925 tubes and head, and a period of 5 hours. Also, material from the tubes was collected through a 15 gauge needle without the application of pressure. Seven-tenths of a m1 samples were collected into each sample tube that contained 0.7 m1 of a Saline—Citrate—KCN solution and read on the Beckman DU at a wavelength of 260 mp. RESULTS + ) with E. coli K-12 arg—T6r--Assuming that E. coli B-3 thy‘ (R+) Conditions for conjugation of E. EQEE B-3 thy- (R was capable of conjugating with E-.£Qli K-12 arg-, it was necessary to see if it would conjugate with E. 39;; K-12 arg_T6r and if §-.£2£l K912 arg-T6r would accept the R factor. A double counterselection of the donor was employed by adding 100 phage per donor to the mixture of donor and recipient cells at the end of the prescribed incubation period and by plating the mixture on Davis synthetic medium without thymine. As can be seen from Tables 1 and 2, the two procedures used for counterselection were effective. The donor cells were eliminated while the phage treated recipient cells were able to produce a population equal to untreated cells. Table 2 also shows that there was no growth of the recipient cells in unsupplemented Davis synthetic medium. This table also shows that there was no evidence of back mutation of the auxotrOphic markers in either the donor or the recipient cells in 107 cells/ml. Table 3 gives a comparison of the resistance to the various drugs of the donor and the recipient prior to the transfer of the R factor into the recipient cells. In addition, it was found that the loss of the R factor by the 7 donor was at a frequency of less than 5 x 10_ Also 35 .HE mom omouoomu muwz mucsoo one .mecoHoo onp mcHucsoo wu0mm£ m>MU N new 0 pm pm omquSUCH mHmB mwumHm .Amuflv mCHchHm HE\ml om Ho Amnev ocHEmnu HE\m1 om Hwano zuHB poucmEoHQQSm EsHme UHumnucmm mH>mQ pcm .Amzv Hmmm ucwHHusc co omDMHm Ucm .owuoHHo .mmEHu m ownmm3 .meSMHuucmo cozy wumB mHHmo wmwne .mmDSCHE Om How 0 um um omumnsucH pom o>onm UwUmHH mEchmmuo mnu mo mwuspHso nuoun ucwHuusc mcH3oum mHHmHucmcomxw on pooom mmz HHmU\mmmnm OOH mo coHumuucwocoo 4 x . x . x . mum I H 00 .1. @0H H v HOHv mOH H v mOH H v moan NH & .H m AOHSDXHE Hqu Hoaumum «Hum HHou am moH x H.v 40H x m mos x H.¢ mos x m.m x A+mv usnu mum Haoo am H3v HOH x m HoH x m mos x m.v A+mv nan“ mum Haoo xm mum has £2 «2 mEmHammHO Umumwuu mmmnm mmmsm oz ill illii .wmmnm we ou oncommmm .H mHQmB 37 .HE mom ompuoomu cmzu oum3 mucsoo mna .mmHCOHoo wnu mcH aucsoo muommfl mmmo m How U um um pmHMQUUCH Conn mum3 mmuMHm .Amuflv wCHchHm HE\m:.om no Amnav wCHahsu HE\m1.om nonuHm SuHB omwcwEmHmmsm EsprE UHuwCDC%m mH>mm com .Ava EdeoE UHDmnucmm mH>mQ .Amzv ummm ucwHuusc co UmpmHm pom .UmusHHo .meHu N ponmm3 .Umm:MHHpcmo .muso: m Mom 0 mm pm QDOHQ ucwHHus: CH c3oum mum3 w>onm UmpmHH mEchmmHo wsfi mos x H.¢ H3v H3v mos x H.¢ Hoaumum NHim HHoo am AOHSDMHE Hqu Hoelmum NHIM HHoo .m. mos x H.v mos x m.v H3v moa x m.m x A+mv -mnp mum Haoo am H3v moa x m.w Hod mos x N.¢ A+mv nan» mum Haou am mu< >36 2m <2 mEchmmHO mflomz .mucwEwuHsvmu £p3onw .N mHQmB 38 Table 3. Response to drugs. Organisms Media IE. coli B-3 thy- (R+) .E. coli K-12 argDT6r NA 4.2 x 108 4.1 x 108 Thy + 50 Cm 1.5 x 108 (101 Thy + 25 Str 1.7 x 108 (101 Thy + 200 Sul 1.7 x 108 <10l Arg + 50 Cm <101 (101 Arg + 25 Str <101 (101 Arg + 200 Sul <101 <101 The cultures and media were the same as reported in Table 2 except that drugs were added in pg/ml as indicated by numbers which precede the drug abbreviations. The incuw bation procedure and counts were also done as in Table 2. 39 the R factor was never spontaneously acquired by the re- cipient cells. As can be seen in Table 4, phenotypic expression of the R factor was evident in the recipient at a frequency of 10‘.3 after a mixture of the donor and the recipient cells was incubated together. It can be noted by comparing Tables 3 and 4 that the resistance to Cm was raised from 50’pg/ml characteristic of the donor to 150 pg/ml in the recipient while the resistance of the recipient to the other two drugs was no different than the donor. The resistance to Cm, Str, and Sul is expressed as the concentration (w/v) of the drug to which most cells were not affected and formed visible colonies. As can also be noted in Table 4, the chromosomal markers of the recipient were not affected by the newly acquired R factor. To show that the R factor was actually transferred to the recipient cell, several colonies, which had developed on the drug containing plates, were picked anfl re—streaked on Davis synthetic medium plus arginine containing either 150 pg/ml Cm, 25 pg/ml Str, or 200 ug/ml Sul. :There was growth of colonies, no matter from which drug containing plate they were picked. It was concluded that they had re- ceived the R factor containing all three drug markers from the donor cells. - - - . . — + _E1trous ac1d—1nduced mutat1ons of E; coEE_B-3 thy (R )1_ Since our objective was to obtain a suppressor mutation which 40 Table 4. Drug resistance ony. coli K912 grguT6r after conjugation with E. coli B-3 thy (R+). Media Organisms/ml NA 4.1 x 108 Arg + 150 Cm 2.2 x 105 Arg + 25 Str 1.9 x 105 Arg + 200 Su1 2.3 x 105 Thy + 50 Cm 9 x 101 Thy + 25 Str 2 1.1 x 102 Thy + 200 Su1 1.1 x 102 All conditions, cultures, and media were described in Table 3 except that in this case the cultures were mixed and allowed to mate for 60 min, the males were then countere selected with T6 phage, and incubated for an additional hour to allow for phenotypic expression of the recipient. 41 would affect the drug resistant markers on the R factor, it was necessary to develop a mutational procedure for this purpose. When the mutagen, nitrous acid, was used as de— scribed by Kaudewitz (1959) to obtain an E._99EE B-3 thy—Su — (R+) mutant, no mutants were isolated. Therefore, R the procedure described in the Material and Methods section was developed. The R factor was not expressed in 12 of 122 potential mutant colonies isolated by the modified nitrous acid and penicillin selection procedure. When these 12 colonies were cultured and crossed with E._29EE K912 arg—T6r, 5 of them were still able to transfer the Cm, Str, and Su1 resistance to the recipient (Table 5). Drops of cultures of these 5 mutants were spotted on nutrient agar that contained various concentrations of the drugs to determine how much resistance they had retained (Table 6). From these results, it was con— cluded that a suppressor mutation had occurred in all 5 of these mutants. Loss of mutants——It was found that when suppressor mutant types were stored for several weeks in Penassay broth in the refrigerator, there was a dramatic decrease in the mu- tants until after a period of about 2 months almost all of the mutants were dead. Therefore, when an inoculum was taken from these stored cultures, more and more_E..ggli B—3 thy— (R+) back mutants were evident. Fortunately, the Table 5. Crosses of_E. coli B—3 thy-[Su mutants with E. coli K912 arg-T6r. 42 R -potent1a1] + (R ) Mutant number Number of surviving recipient cells per ml Arg + 150 Cm Arg + 25 Str Arg + 200 Su1 l 1 19 <10 <101 <10 24 <101 <101 <101 31 <101 <101 <101 42 5.2 x 104 5.5 x 104 5.0 x 104 44 1.3 x 104 1.7 x 104 1.0 x 104 53 <101 <101 (101 59 3.1 x 104 2.8 x 104 2.9 x 104 62 <101 <101 <101 68 <101 <101 <101 71 2.3 x 104 2.7 x 104 2.3 x 104 83 9 x 103 1.1 x 104 1.0 x 104 87 <101 <101 (101 8 In this cross, 3.2 x 108 cells/ml of recipient and 10 cells/ml of the various mutant donors were used. The conditions of mating, the media, incubation procedure, and counts reported were the same as reported in Tables 2, 3, and 4° 43 Table 6. Resistance of E. 39;; B-3 thy-[SuRu—potential] (Rf) mutants. Growth response in drug-supplemented NA Cm Str Su1 Organisms 30* 40* 50* 5* 25* 200* 1000* .E..pp;; B-3 thy. (R+) + + + + + + - Mutant # 42 + — — - _ + + Mutant # 44 + — - _ _ + + Mutant # 59 + — - _ - + + Mutant # 71 + - - - - + + Mutant # 83 + - — — _ + + The organisms listed above were grown in nutrient broth at 37 C for 6 hours, centrifuged, washed 2 times, re— suspended in the same volume, and a drOp was spotted on each of the drug-supplemented nutrient agar plates. Plates were then incubated at 37 C for 3 days before observing for growth. * =‘pg/ml, + = growth, and — = no growth. 44 reselection step described in the next section was carried out before all of the mutant cells were dead. When drops of the mutant cultures were spotted ap— proximately a cm from the parent strain. LE._QQEE B-3 thy_ (R+H , it was noted that growth of the mutant was in— hibited. This inhibition was apparently due to a substance released from the parent strain. Similar inhibition of the R taneous back mutants, E. coli B—3 thy- (R+). From these _E. coli B—3 thy-Su (R+) mutant was caused by the spon— facts and from the fact that there was death in the Penassay cultures stored in the refrigerator, it was concluded that the parent strain as well as the back mutants were producing a colicin-like substance that was lethal to the mutants. Reselection for mutants from refrigerated cultures—— It was necessary to re—select for the mutant types from the refrigerated Penassay cultures before they were completely lost. Once they were re—isolated, it was necessary to re— test them to make certain that they possessed the same level of drug resistance as the mutants isolated originally. This time, however, different growth media were tried to determine if the mutants would survive and remain more stable in one of them than they were in Penassay broth. Nutrient broth was finally selected as the most suitable medium since very few back mutants were found in it after several weeks of storage. For an additional safeguard, the mutants were also 45 stored in saline in the refrigerator. The newly isolated mutants were tested for the presence of a possible suppressor to the R factor and for auxotrophic markers and were found to be similar in all respects to the previously isolated mutants. Characteristics of back mutants--Since back mutants were present in the mutant stocks, it was decided to test several of them to determine if they were truly back mutants to E. 39;; B-3 thy- (R+). However, before this was done, the frequency of back mutants in the nutrient broth mutant stock cultures was determined. It was found that out of 1 x 109 mutants there was 1—16 back mutants. The back mutants were easily detected since, on nutrient agar, they appeared as mature colonies in 24 hours while the mutant colonies required 48 hours to mature. When the mutants were grown on Penassay broth and then plated on nutrient agar, it was found that out of 1 x 109 mutants there were 100-1000 back mutants. Of the 6 back mutants that were tested for their com~ plete reversion to E. EQEE B-3 thy_ (R+), 3 were isolated from plates spread with the nutrient broth cultures while the other 3 were from the Penassay broth cultures. All 6 of these organisms were found to be fully reverted to the parent strain for the characteristics presented in Table 7. The next step was determined if these back mutants were still able to transfer the R factor to the recipient strain 46 Table 7. Growth requirements and fermentation patterns of the donor, the recipient, and a suppressor mutant of E. coli. Growth response _ + _ _ _ . Growth factors thy (R ) arg T6r thy SuR (R+) Arg - + - Thy + — + Additional requirement — - + Fermentation pattern Carbohydrate thy- (R+) arg—T6r thy-SuR- (R+) Lac + + + Mal - + - Mtl + + + Xyl + + + Dex + + + Scr - — - Dul — - - E. Loli B—3 thy (R+ ),_E. cLli K+12 arg T6r and E. Loli B—3_ thy SuR’ (R+ ) for the growth requirement portion were grown, treated, and inoculated as described in Table 6. The incubation procedure and observation for growth were also the same as described in Table 6. The media used was described in Table 2. ,Since E. coli B-3 thy’SuR‘ (R+) did not grow on Thy plates. it was assumed that it had an ad- ditional growth requirement. As for the fermentation pattern, the cultures and treatment of the cultures were the same as above except that 0.1 ml was inoculated into 3 ml of Purple Broth Base (Difco) supplemented with 1% of either lactose (Lac), maltose (Mal), mannitol (Mtl), xylose (Xyl), dextrose (Dex). sucrose (Scr), or dulcitol (Dul) and incubated at 37 C for 2 days before being checked for acid production. 47 (E. 22;; arg-T6r) at the same frequency and bring about the same quantitative response to the drugs as the original donor LE. gglg_B-3 thy- (R+)]° Table 8 shows that the R factor of the back mutant was transferred at a frequency comparable to the parent strain (Table 4). Characteristics of a randomly chosen suppressor mutant——One of the suppressor mutants was arbitrarily chosen and was designated E. coli B—3 thy-SuR- (R+). Since this mutant, as well as the other suppressor mutants, grew much slower than did the parent strain and also gave much smaller colonies on nutrient agar, fermentation tests were performed with the mutants. At the same time, tests were set-up to den termine if the mutant had acquired any growth requirements in addition to those of E. 39;; B—3 thy- (R+)° It can be seen from Table 7 that there was an additional growth requirement for E. coli B—3 thy-SuR— (R+). Also, it can be seen from the table that there was no change in the fermentation pattern of the mutant when it is compared to the parent strain. Test for growth requirement oqu. coli B-3 thy-SuR— (R+)--The tests for the growth requirement of this mutant were carried out on Davis synthetic medium supple- mented with thymine, since there was no reason to believe that the thymine marker had back-mutated to the prototrophic 48 Table 8. Transfer of resistance factor from one of the back mutants of E. coli B—3 thy—Su — (R+) to_E. coli K—12 arg-Ter. R Organisms . Back mutant of Back mutant of thy-SuRf (R+) Med1a thy—SuR- (R+) x arg-T6r NA 5.8 x 108 1.8 x 109 Thy 6.0 x 108 1.7 x 102 Thy + 50 Cm 2.1 x 108 1.9 x 102 Thy + 25 Str 2.6 x 108 1.9 x 102 Thy + 200 Sul 1.9 x 108 1.6 x 102 Arg (101 1.6 x 109 Arg + 150 Cm (119- 5.9 x 105 Arg + 25 Str (119' 5.2 x 105 Arg + 200 Sul (101 6.1 x 105 The back mutant of E. coli thy-Su - (Rf) was plated R alone on selective and non-selective media as well as conju~ gated with E. coli K-12 arg‘T6r as described in Tables 2, 3, and 4. The media and counts were accomplished as described in Tables 2 and 3. 49 condition. It had also been noted that the back mutants of .§°.£2ll B-3 thy—SuR- (R+) were able to grow on Davis syn- thetic medium plus thymine but not on Davis synthetic medium alone. Table 9 shows the growth response around the various crystals. As can be seen, there were several amino acids as well as one pyrimidine that would support at least some growth of this mutant. However, it was very unlikely that this many mutations to auxotrOphic conditions could have + taken place during the treatment of E. 39;; B—3 thy- (R ) with nitrous acid. Thus, a substance was looked for that was either common in the formation of these substances or was present in the pathway going from these substances to another substance. It was noted that the substance which appeared to be common to most of these pathways was tetrahydrofolic acid and its derivatives. Folic acid was the first compound tried, since it was a precursor to the synthesis of tetrahydrofolic acid. However, no growth was observed in the presence of folic acid on the Davis synthetic medium supplemented with thymine. Therefore, if the block was in the tetrahydrofolic acid pathway, it must be at a point after the formation of folic acid. Since tetrahydrofolic acid was unstable, and since the block may be past the formation of tetrahydrofolic ’acid, it was decided to look for a more stable compound that was either further along the pathway or was converted by the mutant into one of these compounds that were further along 50 Table 9. Substances which support the growth of E. coli B—3 - - + thy SuR (R ). Substances Ser Cys Tyr Glt Ala Met Cyt Amount of growth + + + ++ ++ +++ + The growth conditions and treatment of the culture were the same as described in Table 6. However, several plates were then made by adding 0.1 ml of the culture to 3 ml of soft agar and poured onto the surface of plates con- taining Davis synthetic medium supplemented with 20 pg/ml thymine. Crystals of serine (Ser), cysteine (Cys), tyrosine (Tyr), glutamic acid (Glt), alanine (Ala), methionine (Met), and cystosine (Cyt) were placed onto the surface with no more than 4 tests per plate. The plates were then incubated at 37 C for 4 days and the density of growth around the crystals recorded with +++ indicating the best growth. 51 the pathway. Because the Citrovorum Factor and leucovorin both satisfied the second criterion it was decided to try one of them. Since leucovorin was available through Lederle Laboratories, it was used. Leucovorin is a synthetic compound with only about one-half the growth stimulating activity of N5 formyl tetra— hydrofolic acid. The latter compound is a precursor of N10 formyl tetrahydrofolic acid, an intermediary metabolite of the tetrahydrofolic acid pathway. At the same time, it was noted in Guirard and Snell (1962) that serine, methionine, thymine, guanine, and adenine, if added together to synthetic medium, would overcome the requirement for tetrahydrofolic acid. In Davis synthetic medium containing thymine when supplemented with, in one case, serine, methionine, guanine, and adenine; and, in the other case, with 20 pg/ml of leu— covorin, there was, as can be seen in Table 10, growth of the mutant. It should also be noted that there was no growth of E. coli B—3 thy—Su — (R+) on Davis synthetic medium supple~ R mented with the individual amino acids plus thymine nor on the individual purines plus thymine. There was also no growth of the mutant on Davis synthetic medium plus leu- covorin which indicated that thymine was still a requirement for this organism. Different concentrations of leucovorin were used in an attempt to find the minimum concentration that would support growth. Twenty Pg/ml was the minimum that could be used. However, the growth of the mutant on both 52 Table 10. Growth requirements for E. coli B-3 thy-SuR- (R+). Substances Met, Ser, Ade, Leucovorin and Gua Met Ser Ade Gua Growth response + + — - - — The culture and conditions were described in Table 9 with the exception that the plates contained either 20 pg/ml of leucovorin or 20 pg/ml of methionine (Met) plus serine (Ser) plus adenine (Ade) plus guanine (Gua), or 20,pg/ml methionine, or 20,pg/ml serine, or 20,pg/ml adenine, or 20 [pg/ml guanine. 53 these media was slower than the growth of the parent, E. coli B—3 thy- (R+), on Davis synthetic medium plus thymine. Crosses of E. coli B-3 thy-Su _ (R+) and E. coli R 13-3 thy“ (11*) with _1_3_. coli K-12 arg-T6r under almost identi- cal conditions——In order to see if there was any significant difference in the number of R factors transferred from the two different donors, the two crosses were made at the same time,with the recipient in both cases coming from the same tube. There was one variation in the routine mating pro— cedure because of the slower growth rate of E. ggli B—3 thy—Su - R (R+))it was necessary to inoculate the E. coli B—3 thy-Su — (Rf) into nutrient broth 2 hours earlier than R .E..gg;; B—3 thy- (R+). Thus, at the time of mating, both donors were approximately the same concentration. Table ll shows that there was a difference in the number of recipients receiving the R factor, but there was no quantitative difference in the drug resistance between those recipients receiving the R factor from the two different donors. .E- coli B-3 thy_ (Rf) cured of the Rf factor—- Colonies,which grew when replicated onto nutrient agar plates but did not grow on nutrient agar plus lO‘pg/ml of streptomycin, were picked and were grown in nutrient broth. These cultures were then centrifuged, the cells washed and resuspended in saline, and finally a drop of each was spotted on Davis synthetic medium plus thymine supplemented with 54 . o + OH HOHv H.3 x m H HOHv mOH m m HOHv so omH < o VA o o WM HQHv moH x o H mOH m H mOH m a HQHV < . . X .. 5 x . mOH x m H HOHv HoHv N0H m m BOH o n H m com + :H X o X . H + HOHv H.0H m H HOHv RQH m a pm mm a2 HOHV HOHv NoH x m.h Hum m + <2 . x . x . HOHv HoHv. HQHV N0H v m hOH H a so om + :9 X . . x . h . 50H m a HOHv Hodv NOH H n NOH H m so om + as . . . m mOH x n H HOHV HOHv NOH o o mOH x v H as x . x . x . . x . moH m H mOH m H mOH v H mOH m m mOH m H «z m A+mv I smlwnu Hoelmum x HmBImHm Hoelmum x H+mv >£D mflomz H+mc H+mv ssh» mEchmmHO .mCOHUHUcoo HmHHEHm Hmpcs Hoelmum malx HHou .m.:uH3 H+mv -msmumnu mum HHoo .m.6cm H+mv nsnu mum HHoo .m.mo mmmmOHo .HH mHHme 55 .cBOHm o>mn UH503 Houomm m mnu Uw>HmowH pm: LUHSB ucmflmflowu map pom Hocoo may Loon mocflm mUmE THTB mmCHumHQ oc umnu mmumoflocfl SUHSB poms mfl AIV mum£3 ammo wa HE Hmm UTUHOUTH mucsoo Tn“ pom m>MU m How 0 um um Umumnsocfl muwz mmumam Hafl .mcHumE ou HOHHQ umsh monopaso mwmnp mcflumHm >9 vocamuno THTB A my m + I smlwcu pom .Hmelmum .A+mv lazy Umomms mcEDHoo mag Hops: mucsoo one .mmumHm Hmmm 0Hum£uc>w Ham on oopom THTB cHHo>oosmH mo HE\mR.0N ummoxm v pom m mmHQMB CH UTQHHommt mm TEMm msu wumzflprHHHps MHUTE mnu THH£3 g wanme CH pmnfluomwo mm mEmm may mm3 coflummoflcoo How owonQEw THDUTUOHQ one .uofilmum NHIM HHOU .m.©cm A+mv hnu mum HHoo .m Mo mmocp THOMTQ musoz N omwumum mm3 A+mv msmlmnu mum Haoo .m mo musuaoo ms» umnu ammoxm m manme CH confluommp mm mEmm wsu THTB msflpmam Hflmnu pom .mCHmmouo mo TEHD wnn Op HOHHQ ucmEumeu Hflmcu .mmnsuaso on» mo wcoHuHUcoo suzoum mse x . x . 5 mm HOHV voH m H HOHV moa m m HQC H m oom + 4 x . x . H mu HQHv HVoH h H HOHv mOH m m HOHV um mm + m A+mv Imomlhnu HOBImHm x Hofilmum Hofilmnm x A+mv Imnu mflpmz m H+mv u smussu H+mv usgu mEchmmHO UmscHucoo .HH anmB 56 either S‘pg/ml of Cm, or S‘pg/ml of Str, or SO‘yg/ml of Sul. An organism, which did not grow on any of these drug- containing media nor on Davis synthetic medium but did grow on nutrient agar and Davis synthetic medium plus thymine, was used to see if it could transfer drug resistance to E. 39;; KrlZ arg—T6r. The results of this cross showed that no R factors were transferred to the recipient. For more substantial proof that this organism was actually cured, it was crossed with an E._gg;i K212 argnT6r (R+) which had received the R factor from E. coli B-3 thy— (R+). Table 12 shows that the R factor was re—introduced inw to the cured strain but at a much lower frequency than was observed with E. 39;; K+12 arg—T6r as the recipient. The cured E. 99;; B-3 thy- cells which had just received the R factor were then re—streaked on Davis synthetic medium plus thymine that contained the same concentrations of the drugs as they had been selected on and their resistance was found to be stable. Deoxyribonucleic acid extracted fromggells--At each step in the DNA extraction procedure, the precipitate or pellet formed after centrifugation and the supernatant fluid were tested for DNA content with the diphenylamine reagent. In the final step, when the salt concentration of the aqueous lysate was raised to 4 M, most of the DNA, 7,390 pg, was found in the supernatant fluid. However, 200,pg remained in the pellet. 57 Table 12. Cross of an E. coli K-12 arg“T6r (R+) with a cured E. coli B-3 thy‘. Organisms _ r + arg'T6r (R+) _ Media arg T6 (R ) x cured thy' cured thy Arg 4.4 x 108 8.1 x 108 <10l Arg + 150 Cm 1.2 x 108 5.2 x 108 <10l Arg + 25 Str 1.9 x 108 4.9 x 108 (101 Arg + 200 Sul 1.6 x 108 5.0 x 108 <10l Thy (101 7.6 x 108 4.1 x 108 . l 3 l Thy + 50 Cm (10 4.3 x 10 <10 Thy + 25 Str (101 3.6 x 103 (101 Thy + 200 Sul (101 3.5 x 103 (101 2, 3: All conditions employed were as described in Tables and 4 except that the counts under columns headed arg T6r (R+ ) and cured thy were obtained by plating these cultures just prior to mating. 58 Since the DNA of cells was considered to be 3-4% of their dry weight (Luria, 1960), calculations were carried out on the supernatant fluid, referred hereafter to as the ly- sate, to determine the quantity of DNA extracted from the cells. Table 13 shows that 42.0-55.S% was in the lysate. If, however, the DNA makes up only 1% - 3% of the dry weight of the cells (Fuerst and Stent, 1956), then the lysate yielded almost the theoretical quantity of DNA. Since the DNA content can also be determined by using the conversion factor of l O.D. unit at the wavelength of 260 mp = 47 pg of DNA (Ganesan and Lederberg, 1964), it was decided to check the diphenylamine test for accuracy. Table 14 shows that there was almost complete agreement between the DNA content measured by the diphenylamine reagent and by the O.D. conversion factor. Ultraviolet absorption spectrum of lysate--Figure 2 shows the ultraviolet absorption spectrum which was obtained with a 1:6 dilution of the lysate. This spectrum does not follow the spectrum that was obtained with purified DNA but does follow the spectrum of a nucleoprotein. Nucleoproteins are known to have a peak absorption at 260 mp as well as well as having an increase in their absorption below 240 my. This increase in absorption below 240 mp is attributed to the protein portion of the nucleoprotein. 59 Table 13. Percentage of deoxyribonucleic acid extracted from cells. , . _ Wet weight of cells used for extraction ----- =~~2,200 mg Dry weight of cells (20% of wet weight) ------- 440 mg 3~4% of dry weight is DNA content ------------- 13.4 ~ 17.6 mg DNA content of lysate ---------------- , --------- -»7.39 mg Percentage of DNA obtained from cells --------- 42.0 1 55 5% Table 14. Deoxyribonucleic acid content of lysate calculated by O.D. conversion factor. O.D. at 260 mp of 1:6 dilution of 1ysate---_--0.625 O.D. at 260 my for 1 ml of undiluted 1ysate«--3.75 42 ml of lysate x 3.75 O.D. units ---------- 1~157.5 O.D. units Since 1 O.D. unit = 47 pg DNA then 157.5 x 47 ----------------- - ------ .-__17°4025 mg of DNA 60 2.000 1.800. 1.400~> 1.200 1b O.D° 1.000“ 0.800J~ 0.6004 051400" ‘ 0.200« A L 290 240 260 760 230 280 290 mp Figure 2. Ultraviolet absorption spectrum of a 1:6 dilution of the lysate in 0.15 M NaCl plus 0.010 M trisodium citrate plus 0.010 M KCN at pH 7.0. 61 Table 15 presents ratios of the absorption at 260:230:280 mp of Salmon sperm DNA, of the lysate isolated by the author, and of bacterial DNA reported by Marmur (1961). From these ratios, it was tentatively concluded that the lysate was probably a nucleoprotein. Composition of the 1ysate--Since the lysate appeared to be a nucleOprotein, tests were done to determine the pro- portion of protein, DNA, and RNA present. It can be seen in Table 16 that the largest part of the lysate was protein. When the per cent of the nucleic acid per ml of nucleic acid plus protein was calculated, it came out to 16-30% depending upon which protein value, that obtained with the micro-Biuret or with the Lowry test, was used. The calculated per cent of nucleic acid per m1 of extract prepared by the modified Marmur procedure and of the commercially prepared Salmon sperm DNA were 90 and 87%9 respectively. The low percentage of the nucleic acid in the lysate approaches the lower limit, 35%, of a nucleoprotein (Chargaff, 1955). The high percentage of nucleic acid in the other two DNAs is markedly higher than the uppermost limit, 60%, of a nucleoprotein (Chargaff, 1955). These data support the conclusion that the lysate may primarily be a nucleoprotein and that the other DNAs are almost pure DNA. Sodium hydroxide degradation of the lysate——Since sodium hydroxide treatment degrades DNA the same way as does 62 Table 15. Ratios for absorption of DNAs at 260:230:280 mp. O.D. ratios Material 260 g 230 280 Marmur's DNA1 1.0 : 0.45 0.515 Salmon sperm DNA2 1.0 : 0.38 0.513 Lysate 1.0 : 1.55 0.645 1Data obtained from Marmur (1961). 2Nutritional Biochemicals Corp. 63 OOH om mm om H.o.m.zc n30q Conuwz HocHoHo HmHumumE TCHEmaxcoLmHo ustHmlouoHZ m9 campoum >9 mzm >9 «ZQ >3 camuowm HE\@1.CH mmocmumflsm .mzo Eummm coedmm CH ucwmmum omozu on paw wsspoooum HJEHCZ pwHwHUOE m an UTHMHOmH 42m Ca Dcwmoum mmocmumDSm Team mzu mo ucsoem mnu on omHmmEoo mumm>a Tau CH pcwmwum mwocmumnnm .CH THQMH 64 DNase (Hotchkiss, 1957), this treatment was applied to the lysate. Table 17 gives the calculations for determining the amount of degradation of the lysate and shows that there was a 29% increase in the O.D. readings at 260 mu. This indi- cates that the lysate contained double stranded DNA molecules. Attempt to separate thegprotein from the DNA—-A pro” cedure described by Chargaff (1955) for the separation of the histone from the DNA of a nucleohistone was applied to the lysate. However, after adding the ethanol to the lysate that had been raised to 2.6 M sodium chloride, a granular precipitate was obtained as well as granules which adhered to the sides of the test tube. Since most of the material that formed the granules on the side of the tube remained there when the rest of the material was decanted from the tube, it was dissolved in 0.015 M NaCl plus 0.0015 M tri- sodium citrate at pH 7.0. In the meantime, the precipitate that had been decanted was centrifuged at 17,300 x G at 4 C and redissolved in 0.015 M NaCl plus 0.0015 M trisodium citrate at pH 7.0. The absorption of both samples was de- termined at 230, 260, and 280 mp in the DU spectrophotometer. The ratios of these O.D. readings as well as the percent of recovery of the nucleoprotein are given in Table 18. A de- crease in the absorption at 230 mp was an indication that some of the protein was separated from the nucleic acid. Some of the DNA (nucleoprotein) was also lost during the treatment. '65 Table 17. Calculation of the amount of degradation of the lysate with sodium hydroxide. Lysate + Lysate Na0H 260 my reading 0.603 0.731 320 my reading 0.021 -0.005 260 my reading minus 320 mp reading corrects the 260 my reading for interfering substances 0.603 - 0.021 0.582 0.731 — -0.005 0.736 Correction for the NaOH added to the lysate 0.736 X 1.02 = 0.751 Percent increase of O.D. reading brought about by NaOH treatment: 0. _ o 0.582 x 100 — 29A 66 Table 18. Assay by ultraviolet absorption of the ethanol precipitates of the lysate. O.D. ratios Material 260 : 230 : 280 Precipitate on side of test tube 1.0 : 1.18 : 0.63 Precipitate from bottom of test tube 1.0 : 1.16 : 0.64 Percent recovery of nucleoprotein: DNA added to test tube -------------------- 440dpg DNA from side precipitate ————————————————— 142_pg DNA from bottom precipitate --------------- 163 pg DNA from total precipitate ---------------- 305.pg 305 w o 440 x 100 — 69.5% 67 Attempt to remove nucleoprotein by centrifugation—— One ml of the lysate was placed in 3.5 m1 of 0.15 M NaCl plus 0.010 M trisodium citrate plus 0.010 M KCN at pH 7.0 and centrifuged in the SW—39 head of the Spinco model L ultracentrifuge for 2 hours at 114,743 x G. Samples were then collected by pipetting off a series of 0.7 ml samples from the top and placing them in 0.7 m1 of 0.15 M NaCl plus 0.015 M trisodium citrate at pH 7.0. The O.D. of each sample was then ascertained at 240, 260, and 280 my in the DU spectrophotometer. As can be seen from Table 19, there was little nucleoprotein sedimented by this method. The material that was precipitated was washed from the bottom of the tube with distilled water and the O.D. of it was again read at the same wavelengths. Table 19 shows that there was still nucleic acid in this fraction but it was not in as high a proportion as it was in the other fractions collected from the tube. Sucrose density—gradient centrifugation——To see if a molecular weight might be estimated using the method of Burgi and Hershey (1963), one ml of the lysate was laid over a sucrose density-gradient solution contained in a 34 ml SW425 cellulose tube, and 1 ml of DNA extracted by the modi- fied Marmur procedure was laid over the sucrose density- gradient in another tube. The tubes were then centrifuged for 5 hours at 63,581 x G, collected, and read at 260 mp. 68 Table 19. Assay by ultraviolet absorption of 0.7 m1 fractions obtained after centrifugation at 114,743 x G for 2 hours. O.D. Fraction no. 260 280 240 1 0.489 0.315 0.411 2 0.492 0.316 0.408 3 0.470 0.302 0.388 4 0.459 0.298 0.367 5 0.431 0.268 0.358 6 0.449 0.285 0.372 7 0.456 0.296 0.380 Precipitate 0.396 0.348 0.622 Total O.D. = 3.642 Percent recovery: 3.642 x 47 = 171,pg Since 176 pg of DNA were added to the tube A;— x 100 = 97% OH 69 Figure 3 shows that a distinct absorption peak was obtained with the lysate material in those tubes collected from the top of the sucrose density-gradient. Figure 4 shows that two absorption peaks were obtained with the material ex- tracted by the modified Marmur procedure-—one with material from the tOp and the other with material near the bottom of the sucrose density—gradient. Other attempts were made to sediment the lysate further down the sucrose density—gradient by both longer periods of centrifugation and by altering the gradient range, but in each case the lysate was banded near the top of the tube. Sample tubes, indicated by the arrows in Figures 3 and 4, were then read at 230, 260, and 280 mp to determine how these readings compared with those of the original lysate. Table 20 gives the ratios of the absorption at 260:230:280 mp and shows that there was little difference between the material of these tubes and the original lysate. Phenol treatment of the 1ysate--To see if any change in the lysate occurred upon deproteinization, the lysate was treated with phenol by the procedure given by Frankel (1963) and Berns and Thomas (1965). This procedure was employed to prevent an excess of degradation of the material due to shearing forces. The ultraviolet absorption spectrum (Figure 5) shows that the 230 mp peak was reduced considerably. Table 21 shows that the ratios of the absorption at 260:230:280 mp 0.300 70 0.200‘ 0.100‘ Figure 3. 15 26 3b 4b 50 Tube number Absorption at 260 m of 0.7 ml samples collected from a 5—30% sucrose density—gradient after the sedimentation of the lysate. 0.500 4 0.400 0 O.D. 0.300 “ 0.200 - 0.100 ” 0 10 20 3b 40 Tube number Figure 4. Absorption at 260 mp of 0.7 ml samples collected from a 5-30% sucrose density-gradient after the sedimentation of the DNA extracted by a modified Marmur procedure. 72 Table 20. Ratios for absorption at 230, 260, and 280 mp of material from tubes indicated by arrows in Figures 3 and.4. ___ fi— Figure 2 O.D. ratios Tube number 260 : 230 : 280 34- 1.0 : 1.54 : 0.62 39 1.0 : 1.49 : 0.62 42 1.0 : 1.56 : 0.62 44 1.0 : 1.45 : 0.67 Lysate* 1.0 : 1.55 - 0.645 Figure 3 O.D. ratios Tube number 260 : 230 2 280 6 1.0 : 0.47 : 0.52 41 1.0 : 0.46 : 0.51 44 1.0 : 0.46 : 0.52 DNA isolated by a modified Marmur procedure* 1.0 : 0.45 : 0.515 * = 260:230:280 mp ratios of material before centrifugation. 0.800 73 0 . 7000 V 0.600‘ 0.500“ O.D. T 0.4004 0.300" 0.200% 0.100‘ b {D p 290 Figure 5. 240 250 26% 270 280 290 300 mp Ultraviolet absorption spectrum of a 1:2 dilution of the phenol treated lysate in 0.15 M NaCl plus, 0.010 M trisodium citrate plus 0.010 M KCN at pH 7.0. 74 Table 21. Comparison of ratios for absorption at 230, 260, and 280 mp of phenol-treated lysate with other DNAs. O.D. ratios Material 260 : 230 : 280 Marmur's DNA1 1.0 : 0.45 : 0.515 Salmon sperm DNA2 1.0 : 0.38 : 0.513 Lysate 1.0 : 1.55 : 0.645 Phenolized Lysate 1.0 : 0.535 : 0.578 1Data obtained from Marmur (1961). 2Nutritional Biochemicals Corp. 75 were now nearer to those that were obtained with the material isolated by the modified Marmur procedure. The protein ascertained by the Lowry ggugl. (1951) test and by the micro- Biuret test was lower than that of the lysate (Table 22) but was still higher than the protein associated with the DNA isolated by the modified Marmur procedure. This material was then sedimented through a sucrose density—gradient under the same conditions as used for the lysate to see if there was any difference in the sedimentation patterns. Again, as can be seen by Figure 6, the material remained near the top of the tube. Arrows on the graph indi- cate the tubes containing samples that were assayed for their ratios of absorption at 230, 260, and 280 mp and as can be seen in Table 23, there was again no difference when compared to those of the phenolized lysate. 76 om CH mH mm wuspmoonm HCEHmz omHmHmoa an «2n om mm 6H 6.6H mummmH emNHHocmnm oHH HmH.H mHm o.Ha mummmH ponumz porno: ponumz >H30H pozpmz HOCHUHO HMHprmz mcHsmHHcmhmHn Hmuaam10H0Hz an chpoua an «zm an 58 an 5305. HE\mC CH mmocmuwnsm .muspmuoum HCEHmz pmHMHpoE msu >9 pouMHowH «29 may CH pCm mpmmmH mnu CH uComon mmonu ou pmummeoo mm mummmH UmpmmuynHOCwnm map CH qummHm mooCmquCm .NN wHQmB 77 0.200 0.100.. f0 20 30 40 Tube number Figure 6. Absorption at 260 mp of 0.7 m1 samples collected from a 5—30% sucrose density-gradient after the sedimentation of the phenol treated lysate. 78 Table 23. Ratios for absorption at 230, 260, and 280 mp of material from tubes indicated by arrows in Figure 6. O.D. ratios Tube number 260 : 230 : 280 42 1.0 : 0.540 : 0.575 44 1.0 : 0.543 : 0.581 45 1.0 : 0.571 : 0.595 Phenol—treated lysate* 1.0 : 0.535 : 0.578 * = 260:230:280 mp ratios of material before centrifugation. DISCUSSION Four main observations of the R factor seem worthy of further discussion. These observations are: (1) counter- selection of the donor with T6 phage, (2) higher colony counts on the same dilution of E._ggll B—3 thy- (R+) on nutrient agar than on drug-containing media, (3) an apparent loss of drug resistance by the R factor and of a colicin- 1ike substance caused by a suppressor mutation, and (4) a mutation produced by nitrous acid treatment of E, 29;; B-3 thy- (R+) causing the simultaneous requirement for tetrahydro- folic acid or a tetrahydrofolic acid derivative and the loss of drug resistance and a colicin—like substance. Watanabe (1963) reported that if a mixed culture of the donor strain containing the R factor and the recipient were plated on drug-containing media, the donor cells re- duced the active concentration of the drug. Thus, in order to prevent the destruction of the drugs by the donor, Watanabe suggested the use of a T6 sensitive donor and a T6 resistant recipient in all crosses and counterselection of the donor with T6 phage. I found, however, that, whether the donor strain used in this study was destroyed or not, there was no apparent change in the colony count of the recipient. Never- theless, T6 counterselection was utilized in all mating 79 80 experiments except the one in which cured §-.£Qll B-3 thy- organisms were used. In order to attempt to explain the other three obser— vations under one hypothesis, it is necessary to use two known facts and one assumption. The first known fact is that there is evidence that the R factor does become closely associated with the chromosome (Watanabe, 1963). The second known fact is that drug resistant mutations on the chromosome are associated with slow growth of the mutant in drug- containing media (Watanabe, 1963). The assumption is that phenotypic expression of drug resistance by the R factor de- pends upon its association with the chromosome. Thus, the second observation, more colonies on nutrient agar than on drug—containing media, could be ex— plained by the above hypothesis where the non-expressed cells would be considered as having their R factors in the autono— mous condition. When the R factors were in the autonomous state, their only known function would be that of reproduction. Therefore, until a "decision" is made by the R factor, analo- gous to temperate phage, to become associated with the chromosome, there is no phenotypic expression by the host cell as far as drug-resistance is concerned. What determines this decision is not known but must be a critical, rather delicate intracellular environmental alteration. As with temperate phage, this association of the R factor with the chromosome results in the development of 81 immunity to other R factors (Watanabe, 1963). It has been postulated that these events in temperate phage are caused by a repressor substance that it produces (Kaiser and Jacob, 1957). The R factor may thus have a locusresponsible for the production of a repressor. If this is true, mutants of the R factor may be produced in which the R factor never associates with the chromosome and thus never produces im- munity. In support of such mutant R factors are reports that some R factors are not transferable by conjugation (Watanabe, 1963). Further evidence for the association of the R factor with the chromosome was provided by the difficulty of ob- taining a'E.‘gg;i B-3 thy- (Rf) that had spontaneously lost its R factor or was cured by acridine dyes. Watanabe (1963) reported similar findings and attributed them to the inte- gration of the R factor with the chromosome. Hirota (1960) also showed that acridine treatment of bacteria which con- tained the F particle eliminated the particle unless it had become integrated into the chromosome. The third observation, the suppressor mutation of the R factor, can be explained by the hypothesis proposed by Crick._§._;. (1961). This hypothesis, that has been tested quite thoroughly, states that a suppressor mutation brings about the addition or deletion of base-pairs to re—establish a wild-type triplet code beyond the point of the mutation. The inability to obtain a cured suppressed mutant is 82 indicative that there is chromosomal association of the R factor in this strain. The back-mutation frequency shows that the hypothesis proposed by Crick g; 51- may be the mechanism of suppreSsion operating in this organism. Im- plicit in this hypothesis is the requirement that only closely linked sites would be involved in this type of sup- pression. Therefore, the tetrahydrofolic acid's or one of its derivative's genetic site, the suppressor's genetic site, the insertion or attachment site of the R factor and of the colicinogenic factor must be closely linked on the chromo— some. Thus, the fourth observation is accounted for. The attachment or insertion site of the R factor to the chromosome must be unstable (Watanabe, 1963) since it is readily transferred by the suppressed Inutant to E. 29;; K912 arg-T6r. The drug resistance then acquired by the E. 39;; Ke12 arg-T6r cells that have received the R factor is as high as if it had received the R factor from the un- suppressed strain 0f.§-.£Qll B-3 thy- (R+). Since neither the suppressor nor the tetrahydrofolic acid (or the deriva— tive of tetrahydrofolic acid) markers are transferred to E. ggli K+12 arg_T6r, these markers must remain on the chromo- some. Furthermore, this indicates that there is no chromo- somal transfer accompanying the R factor. Although these explanations of the last three obser- vations are speculative, theyrequire fewer 22 Egg assumptions than would be necessary if the transfer RNA suppressor 83 mutation hypothesis (Benzer and Champe, 1961; and Crawford and Yanofsky, 1959) was invoked. The fact that the suppressor mutation had no effect on the sulfathiazole marker of the R factor was anticipated because of the presence of the tetrahydrofolic acid (or the derivative of tetrahydrofolic acid) marker mutation. Since sulfonamides inhibit a metabolic step prior to the formation of folic acid (Lascelles and WCods, 1952), the precursor to tetrahydrofolic acid, there would be no inhibition of a mutant that requires a product beyond this step. The sup- pressor mutant is of this type. The metabolic block pos- sessed by this suppressor mutant appears to be located either just prior to the formation of tetrahydrofolic acid or at a step leading to one of the derivatives of tetrahydro- folic acid. Additional experiments with mutants of the R factor must be done to elucidate whether the R factor is maintained in the cell as is prophage, whether it is expressed only in the chromosomal condition, and how its cytOplasmic or chromo- somal existence is regulated. The R factor probably contains about 5 x 104 nucleo- tide pairs; therefore, in the autonomous state, it should be easily separated from the chromosomal DNA molecule. Whereas, when the R factor is associated with the chromosome, there would not be a significant increase in magnitude to enable one to separate it from the non—R containing chromosome. 84 Thus, an extraction procedure to separate the chromosomal from the episomal DNA should provide evidence about the location of the R factor of the non—suppressed and suppressed mutant. Similarly, the attached or autonomous state of the R factor could be studied with cells grown and harvested in the presence and absence of drugs. The procedure which appeared best for the isolation of "unaltered" DNA was to gradually lyse the cells using a modified method of that utilized by Cairns (1963). The sucrose concentration was, thus, lowered by gradually de- creasing its concentration to a point of extinction in order to allow for the slow leakage of the cellular contents. This procedure would therefore prevent shearing forces from being applied to the DNA by the instantaneous lysis of the cells. This entire procedure was done in the presence of 0.01 M KCN to prevent the synthesis of DNA and to destroy any con- taminating organisms. After RNase treatment of the lysate, the NaCl concen— tration was gradually raised to 4 M in order to free the DNA from the non-specifically absorbed protein and to precipitate the freed protein. The salt concentration was then lowered to 0.15 M at which point the DNA should have been in its most insoluble and most stable molecular form. Sodium lauryl sulfate (duponol) had been used originally for the lysis of the cells, since it not only lyses the cells but denatures some proteins and suppresses 85 DNase activity (Marmur, 1961). The use of duponol appears to be the preferred procedure to lyse the cells when the DNA from them is to be sedimented in a sucrose density—gradient. Lysozyme, on the other hand, would probably leave the DNA complexed with basic proteins and protamines and thus less liable to breakage by turbulence as was pointed out by Cairns (l963b). Also, Hanawalt and Ray (1964) found that, when they attempted to lyse cells with duponol instead of lysozyme, they were unsuccessful in obtaining an isolated DNA of isotopically labelled replicating material in a cesium chloride density-gradient. Thus, lysozyme was used in preference to duponol to lyse the cells. It appears that the DNA isolated by this procedure is double-stranded and associated with protein but considerably degraded and/or altered in its sedimentation prOperties in the sucrose density-gradient. To explain the latter obser— vation, the following arguments are offered. First, and the most likely, an endonuclease has acted upon the nucleoprotein and has enzymatically degraded it into small pieces that, as Burgi and Hershey (1961) showed, would sediment near the top of the sucrose density-gradient tube. This endonuclease would have had to come from the cells, since there was no endonuclease activity found in the RNase preparation. This endonuclease would also have to be different from endonu— cleases I, II, and micrococcal, since the pH utilized throughout the isolation procedure together with the EDTA 86 should have blocked its reaction with DNA (Bhagavan and Atchley, 1965). Also, this endonuclease would have to be one which could cleave the DNA molecule at those places where a specific sequence of nucleotide residues were found and these residues would have to be evenly spaced throughout the molecule to give the sedimentation pattern obtained in the sucrose density—gradient. Second, an enzyme has broken the protein ”linkers” (Cairns, 1962) that could be located between the different genes of the chromosome and thereby produce segments that have a relatively uniform molecular weight. On the basis of the calculations presented in Table 24, this is probably not the case unless these small particles possess an entirely different aggregating property or charge than the larger DNA molecules. If the latter is true, then the sedimentation pattern in the sucrose density-gradient of the nucleoprotein isolated in this study could not be used to determine its molecular weight with any accuraCy. Third, if there is really no degradation of the molecule during the isolation of the nucleoprotein, there must have either been a change in the aggregating property or the charge of the nucleoprotein so that it no longer be— haves the same in the sucrose density—gradient as do the DNAs isolated by other procedures. From some work that has been done recently, it appears that whatever has happened to the DNA upon its isolation must have taken place during its extraction from the cell. 87 Table 24. Molecular weight of smallest gene as compared to molecular weight of nucleOprotein. Minimum size of protein produced by a gene -------------------------------- 250 amino acids If consider 6 nucleotides/amino acid ——————— 1,500 nucleotides (double stranded DNA) per protein If average molecular weight of ————————————— 4.5 x 105 molecu- nucleotide is taken as 300 lar weight of gene Using the equation D2/D1 = (Mz/Ml)0'35 that was described by Bergi and Hershey (1963) where D is the distance in cm that the material sedimented in the gradient and M is the molecular weight, the calculations for the molecular weight of the nucleoprotein are: 3 x 107 — molecular weight of DNA extracted by Marmur's procedure (1961) 4.96 cm — distance which DNA extracted by the modified Marmur procedure sedimented in the sucrose density—gradient. 0.59 cm - distance which the nucleoprotein sedimented in the sucrose density-gradient. 7 4.96 _ ( 3 x 10 ) 0.35 0.59 “ M1 M1 = 6.97 x 104 which is equivalent to about 232 nucleotides. 88 When the extraction procedure was carried out at 4 C, followed by RNase treatment, and finally treated with phenol (Berns and Thomas, 1965), the same sedimentation pattern was observed in the sucrose density-gradient. Thus, additional work will have to be done to (l) elucidate which of the above explanations, if any, are responsible for the behavior of this nucleoprotein, (2) determine how to isolate and characterize the nucleoprotein so that its molecular weight can be determined accurately, and (3) determine how the R factor is associated with the chromosome. SUMMARY A mutant of E. 22;; B—3 thy- (R+), induced by nitrous acid, was isolated which had a block at some step either in the conversion of folic acid to tetrahydrofolic acid or in the formation of one of the derivatives of tetra- hydrofolic acid. This mutant was also found to possess a suppressor action upon two of the drug resistant loci of the R factor and thus reduced the drug concentration to which this organism was susceptible. The parent strain of this mutant was resistant to 50‘pg/ml of Cm and 25‘pg/ml of Str while the mutant was resistant to only 30 pg/ml of Cm and less than S‘pg/ml of Str. However, upon the passage of the R factor from the mutant into the recipient cell, E. 29;; K—12 arg—T6r, the resistance to Cm and Str was found to be the same as when the non-mutated E. ggli B—3 thy_ (Rf) was used as the donor. Thus, it appeared that a mutant oqu. .ggli B—3 thy- (R+) now possesses a suppressor mutation that was located on a chromosomal locus that controlled the formation of tetrahydrofolic acid or one of its derivatives. The fact that this mutant was a point mutation was shown by its low back mutation frequency. The attempts to obtain separation of the episome from the chromosome were not successful. However, a 89 90 nucleoprotein was isolated and characterized. It possessed 16-30% nucleic acid, contained double stranded DNA, and was not sedimented appreciably in sucrose density-gradients of various concentration ranges. 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