I! l 1) i HI HI I 403—; I mcooo H l l HOST-CONTROLLED MODIFKATION OF AN {NTERMEDIATE SEX FACTOR SN BACTERIAL CONJUGATION Thesis for fhe Degree of M. S. MECHIGAN STATE UNIVERSITY Barry Alan Friedman 1965 THESIS LIBRAR 1’ Michigan 5L. cc ‘ University ABSTRACT HOST-CONTROLLED MODIFICATION OF AN INTERMEDIATE SEX FACTOR IN BACTERIAL CONJUGATION by Barry Alan Friedman Bacterial conjugation was performed via three different methods: millipore, centrifuge, and flask, to determine the efficiency of transfer of a F-lac+ particle by each method and to observe the occurrence of host—controlled modification. The efficiency of transfer was found to vary with the method as well as with the organisms. The superior method was the millipore method, while the centrifuge and flask methods usually produced similar results. Restriction was found when Salmonella pullorum was used as both a donor and recipient; a greater restriction was noted in the donor state of interstrain crosses. Escherichia 32;; BB also donated the F-lac+ without difficulty to other K-12 and BB recipients, but was restricted by §, pullorum, HOST-CONTROLLED MODIFICATION OF AN INTERMEDIATE SEX FACTOR IN BACTERIAL CONJUGATION By Barry Alan Friedman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1966 To Mom, Dad, Jan, and Brian 11 ACKNOWLEDGMENTS I wish to thank Dr. Delbert E. Schoenhard for his patience, understanding, and consideration throughout the course of this study. The interest he has shown through continuous discussions have helped to give meaning to this thesis. I would also like to thank Mr. Otis W. Godfrey and~ Mr. Bruce C. Kline for their suggestions and hours of discussion that are reflected in this thesis. During the course of this study, I was supported in part by a Graduate Research Assistantship. B.A.F. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . iii LIST OF TABLES O O 0 O O O O O O O O O 0 v LIST OF FIGURES . . . . . . . . . . . . . vi Chapter I. INTRODUCTION . . . . . . . . . . . II. LITERATURE REVIEW . . . . . . . . . . l 2 Conjugation . . . . . . . . . . . 2 Host-controlled Modification . . . . . 3 8 8 III. MATERIALS AND METHODS . . . . . . . . Cultures . . . Media 0 . I O O O 0 0 O O 0 O O 0 8 Reagents . . . . . . . . . . . . 12 Mutation Procedur . . . . . . . . . 13- Mating Procedure . . . . . . . . . l3 Flask method . . . . . . . . . . 1A Centrifuge method . . . . . . . . 15 Millipore method . . . . . . . . 15 Iv. RESULTS 0 O O O O O 0 0 O 0 O 0 0 16 Series I . . . . . . . . . . . . 16 Series II . . . . . . . . . . . . 21 series III 0 O O O O O O O O O O 28 v. DISCUSSION 0 O O O O O O O O O O 0 3“ Methods of Conjugation . . . . . . . 3N Host-controlled Modification . . . . . 36 VI 0 SUMMARY 0 o o o o o o o o o o o o 3 9 VII. BIBLIOGRAPHY . . . . . . . . . . . “0 iv TABLE 1. 2. 3. A. LIST OF TABLES Page Bacterial strains . . . . . . . . . . 9 Variations of strains and mode of production . 10 Media used and purpose of each . . . . . 11 Frequency of transfer of F—lac+ with restricting and nonrestricting recipients in Series I . . 20 Frequency of transfer of F-lac+ with restricting, and nonrestricting recipients in Series II . 27 Frequency of transfer of F-lac+ from S. ullorum 35 F-laci' to E. coli AB113 lac“ and ET coETI‘EE" lac- . . .—. C O O O C ' O T O O O 32 Frequency of transfer of F-lac+ with restricting and nonrestricting recipients in Series III . 33 LIST OF FIGURES FIGURE Page 1. Summary of the transfer of F-lac+ in the con- trol crosses of Series I . . . . . . . . l7 2. Fre uency of transfer of F—lac+ from E. coli 3. Frequency of transfer of F-lac+ from E. coli A3113 F-lac"' to E. coli A8266 lac-. . . . . 19 A. Summary of the transfer of-F-lac+ in.Series II o o o o o c o o o o o O O O o 22 5. Freguency of transfer of F-lac+ from E, coli 75 F‘lac to E0 9—0—11 BB 130-. c o o o o 23 6. Frequency of transfer of F-lac+ from E, coli BB F-lac to E, coli BB lac“. . . . . . . 2A 7. Frequency of transfer of F—lac+ from E, coli BB F-lac to E, coli AB113 lac“. . . . . . 25 8. Frequency of transfer of F—lac+ from E. coli 9. Summary of transer of F-lac+ in Series III . . 29 10. Frequency of transfer of F-lac+ from E, coli AB785 F-lac"' to §_. pullorum 35 lac' . """'. . . 3o 11. Frequenc of transfer of F-lac+ from S. pullorum 35 F-lac¥ to S, pullorum 35 lac’ . . . . . vi 31 INTRODUCTION The study of host-controlled modification has of late gained momentum due to the emphasis placed on molecular biology. Because both phage and bacterial de-~ oxyribonucleic acid (DNA) have been found to'gain new nonheritable properties without altering their observed genetic content, the basis of the phenomenon seems to lie on a molecular level. Host-contrdled modification has two components: modification and restriction. Modification is controlled by the host cell; it acts directly on the DNA and alters the base sequence or more likely certain portions of the base sequence. When the phage or chromosomal material of the bacteria is then transferred to another cell, it can be identified either as "self" or "nonself." If it is identified as "nonself," restriction occurs and the incom- ing DNA is degraded. If the material is recognized as "self," no breakdown occurs and the DNA can successfully deve10p within the cell. The study involving host—controlled modification was undertaken to determine whether these properties might be extended to various strains of Escherichia coli and Salmon- ella pullorum. F-lac+ was used to test for host-controlled modification and three different methods were employed to analyze the efficiency of transfer. 1 LITERATURE REVIEW Conjugation The process of bacterial conjugation is a unidirec- tional transfer of genetic material from donor to recipient cells in which contact is required (Hayes, 196A). The discovery of this kind of chromosomal transfer was made by Lederberg and Tatum (1946, a,b). The donor state of a cell is imparted by genetic elements known alternately as fertility factors, sex factors, or F-factors and these elements are known collec- tively as F+ (Hayes, 1953 a, b). The F+ is one example of an episome (Jacob and Wollman, 1961); it may exist auto- nomously in the cytoplasm and replicate independently of the chromosome, or it may be integrated on the bacterial chromosome and replicate with it. While in the cytoplasm, replication occurs faster than that of the bacterial chromosome (de Haan and Stouthamer, 1963), but appears to stabilize with time. The integration of the F+ on the chromosome gives rise to what is known as high frequency of recombination (Hfr) donor cells (Hayes, 1953 b). Genetic markers transfer at a rate 1000 times greater in the Hfr than in the F+ state. The Hfr also determines the location of the origin and the resultant order in which the chromosome will transfer (Jacob and Wollman, 1958). Rarely is the Hfr itself trans- ferred. The F+, in contrast, promotes its own transfer, but not that of the chromosome. The sex factor is not re- moved by treatment with acridine dyes (curing) from Hfr strains, but has been removed from strains harboring the F+ (Hirota, 1960; Watanabe and Fukasawa, 1961). In addition to the F+ and Hfr strains of bacteria there exists a third type that is of an intermediate nature (Adelberg and Burns, 1959, 1960). In this case the sex factor is attached to a fragment of the bacterial chromo- some-the length varying from one to several markers. The resultant is known as a F-merogenote (Clark and Adelberg, 1962) or F—prime (F'), and transfer has been referred to as sexduction, F—duction (Jacob and Wollman, 1961), or F- mediated transduction. The F—prime fragment is similar to the F+ in that it behaves as an episome. The F—prime may recombine with the bacterial chromosome if homology is present; otherwise it will only multiply in the cytoplasm. HostfcontrolledgModificatign Host-controlled modification was discovered early in the 1950's when it was found that certain phages could gain new ~nonheritable prOperties without altering their genetic content when passed through a host bacterial strain (Luria Iand Human, 1952; Bertani and Weigle, 1953). Subsequent passage in the same strain resulted in only a minor, if any, decrease in efficiency of plating. However, passage into a second host strain resulted in symmetry or asymmetry. Symmetry refers to the restriction of a phage propagated in one strain from multiplying in a second, and those phage propagated in the second from multiplying in the first. Asymmetry refers to the restriction of a phage propagated in one strain from multiplying in a second, but phage propagated in the second are capable of multi- plying in both. In neither case must the results be quantitative (Arber and Dussoix, 1962). Work by Arber and Dussoix (1962) has shown that ;DNA; carries the host specificity. Bacteria infected with labelled, restricted phage were found to degrade the phage as observed by the appearance of radioactive breakdown products. Experiments conducted with conserved, semiconserved, and newly synthesized DNA gave evidence that only the newly sythesized DNA of the phage carried no host specificity for its former bacterial host. Host-controlled modification appears to be under genetic control. Bacterial mutants have been isolated that while no longer restrictive (r’), still carry out modification (mf); others have been isolated that do not restrict or modify (r’ m‘). Both types are in contrast to the wild type (r+ m+) (Glover et al., 1963). Some have been found that show intermediate modification activity. These include as!» . 5‘“. .__-_ —_ temperature-sensitive mutants giving good modification at low temperatures but little or no modification at high temperatures, and streptomycin mutants (Lederberg, 1957) that affect both restriction and modification. The role of methylation is being investigated as the biological mechanism for host—controlled modification. Arber (1965) deprived methionine-requiring auxotrophs of E, ggli_K-l2 of methionine while the vegetative phage were being replicated. Methionine was then added to permit phage synthesis and maturation. The early, mature phage were found lacking in host specificity. Klein and Sauer- bier (1965) found that host-controlled modification of T1 DNA by lysogenic host bacteria involves methylation of the DNA which can be suppressed by simultaneous infection with T3. Direct evidence is lacking for host-controlled modi- fication at the present time. Gold and Hurwitz (1964 a, b) have isolated a number of methylating enzymes and have meas- ured the uptake of labelled methyl groups using both enzymes and DNA from the same and different strains. Ledinko (196A) found that phage lambda contained equal amounts of 5—methy1cytosine when propagated in strains of E, 2211 B, C, K, or K(Pl). Thus, if host-controlled modification in- volves methylation, it appears to be determined by only a fraction of the bases methylated, presumably in a few specific sequences (Stacey, 1965). Direct evidence has been obtained with the T-even phages regarding the role of uridine diphosphoglucose (UDPG). Phage released from mutants of salmonellae and E. gglilB/A deficient for the capacity to synthesize UDPG were found to be restricted in E, 331; B, but not in shigellae. These experiments suggest the presence of a nuclease in E. 3211 B, but absence in shigellae which can prevent the development of phage not carrying the prescribed amount of glucose. Host-controlled modification may be demonstrated via conjugation involving chromosomal DNA, as well as F+ and F-prime episomes. With chromosomal DNA the linkage between chromosomal markers was found to be reduced in restrictive crosses (Boyer, 196A; Pittard, 196A; Colson gg_§l., 1965; Hoekstra and de Haan, 1965). They also reported that the locus of restriction was closely linked to the threonine locus in E. 3311 K-l2 (Boyer, 1964; Pittard, 196A; Colson et al., 1965) and E. coli B (Hoekstra and de Haan, 1965). The results of restriction have also been reported with F- gal (Hoekstra and de Haan, 1965), F-lac, F+ (Boyer, 196A), and RTF (Arber and Morse, l965)--the efficiency depending upon the system and episome utilized. The passage of bacterial DNA may be hindered by in- efficient copulation. It may also be hampered by the pre- vailing physiological conditions. In addition, nonhomology of donor and recipient DNA may be a reason for unsuccessful exchange of genetic material between bacterial strains which are not closely related. However, transfer of episomes, such as F-prime, which can express themselves without in- tegration into the chromosome should not be greatly affected by nonhomology, thus leaving the task to host-controlled modification (Arber and. Morse, 1965). MATERIAL AND METHODS Cultures The bacterial strains utilized and those genetic characteristics which are pertinent to this study are listed in Table I. Table II lists all variations of the above strains and includes the mode of production. 142912 The media used during the course of this study are listed in Table III. Triphenyl tetrazolium chloride agar (TTZ agar), a medium discovered by Lederberg (1948), was prepared by adding 23 g of nutrient agar (Difco) to 1000 ml of water and steaming until dissolved. To this was then added 50 mg/liter of 2,3,5. -triphenyl-2H—tetrazolium chloride (Eastman Organic Chemicals) and 1.0% (10 g) lactose (Pfanstiehl). The medium was autoclaved at 121 C for 15 minutes and then supplemented with dihydro-streptomycin sulfate (Squibb) to give a final concentration of 200 ug /ml. Triphenyl tetrazolium chloride synthetic agar was pre— pared in the same manner as a modification of eosin methylene) blue synthetic agar (Lederberg, l950)4-the dye constituting the only change. Supplements for the growth of E. 321; AB266 were also added. .pcmpmfimoa u n mo>Hpfimcmn u m mooosoona no uoNHHde no: u I moooBoOAQ no ooNHHde u + mafiomEOBQonpm u ppm mapflaflpoa nos mcoaposooam oaooca u Una momOth u HAN MHOpficcoE u Hus monouoma u and monouowamw u Ham momocfinmnm u can mocacoonzu u use mosHEman u as» mocfiaoaa u 099 moQHGOHnuoE use mocaosma u Boa mocfivfiumfin u was mocfioammo u who ”maonsmm can mcofipmfi>onnooom one o» snowmoooc mm cocoa mm: Caomsoudonpmc confidv Ion no mafismufi> 62m mvfiom ocfiEm cofipmuaaflps omOpomH "muGBGHoEooon mo coapoouon finance o>duooaom coapmNHHfips omouomH umpcwcfinsooon mo coapoopmo Aeam Neev name onumnnaam monaoano EBHHonmppoB Hzconqfine Aaeev name meaaoano EzHHoumnuoe Hmcondfine finance HmfipconoMMHa oomfim sz Aooeaov nmwoq mucosoaaadm omoanzm condom ssfiooz c.20mo mo omoansd can con: BHUoZII.m mqm¢e 12 Sodium Succinate 5.0 g NaCl 1.0 g (NHu)2SOu 5.0 g K2HPOu 2.0 g Lactose (Pfanstiehl) 10.0 g Bacto-agar (Difco) 15.0 g 2,3,5—triphenyl-2H— tetrazolium chloride 50.0 mg Distilled water 1000.0 ml The amino acid supplments were added to give a final con- centration of 20 ug/ml; they include leucine, proline, and threonine. Thiamine HCl was added to give a final con- centration of 5 ug/ml. Reagents The production of indole was detected in SIM agar stabs after 2“ hours of incubation by overlaying the medium with 0.5 ml chloroform followed by 0.5 ml Kovac's reagent. A deep red hue occurred in the chloroform layer when indole was present. Kovac's Reagent Amyl alcohol 75.0 ml Hydrochloric acid (12 N) 25.0 ml p-Dimethylaminobenzalde— hyde 5.0 g l3 Mutation Procedure E, 3211 BB, taken from a nutrient agar slant, was inoculated into 10 ml of penassay broth (Difco) and incu- bated at 37 C until the culture was in the logarithmic phase (10 hours). One ml of the culture was then transferred to 9 m1 of fresh medium and incubated for an additional 2 hours. After the 2 hour period the cells were pelleted by centrifugation and resuspended to a concentration of approx- imately l x 108 cells/ml in penassay broth. Five ml were removed and irradiated for 80 seconds at a distance of 13 3/4" in a Petri dish that was placed upon a Mag-Mix (Precision Scientific) and rotated with a magnetic stirrer. This irradiation produced a 99% kill. A 30 watt, 35 inch long General Electric Germicidal Lamp (G 30T8) was employed for the purpose of irradiation. The bacteria were then incubated in the dark for 6 hours. After the 6 hour incubation period, the culture was plated either on nutrient agar or nutrient agar gradient plates supplemented with 200 ug/ml streptomycin. Two to eleven colonies were found on each plate. Selected colonies were subcultured four times on TTZ—str to confirm the re— sistance to streptomycin and to determine if the ultra- violet treatment had affected the lactose phenotype. Mating‘Procedure Three different methods were utilized to test for host— controlled modification. In each case the bacteria were 124 taken from a nutrient agar slant, inoculated into 10 ml of penassay broth, and grown until in the logarithmic phase, i.e. 11 hours for E. 33;; and 10 hours for E3 pullorum. A 10 ml amount of each suspension was added to 90 ml of penassay broth and incubated for an additional 2 hours (E. BREE) or 3 hours (E. pullorum) to insure logarithmic growth. The cells were then spun on a centrifuge (Servall) at 12,100 x g for 15 minutes and resuspended in penassay broth to give 10 males to 1 female or approximately 1 x 109 males/ml to 1 x 108 females/ml (Echols, 1963). (See indi- vidual tables and graphs for exact ratios as well as varia- tions in procedure). Flask Method--The cell suspensions were prewarmed for 10 minutes with a temperature block (Chemical Rubber Co.) at 37 C and 4 ml of each were placed in a prewarmed 125 ml Erlenmeyer flask. The flask was rotated for 5 minutes to insure maximum contact and then allowed to remain motion- less until each sample was drawn. Samples were drawn at 15, 30, 60, and 120 minutes. One ml was added to a 20 x 150 mm test tube and agitated on a Vortex Junior (Scientific Industries, Inc.) mixer for one minute (Pittard and Adelberg, 1964). Immediately following this, the sample was diluted 104 and placed in an ice bath until usage. Dilutions were then prepared to give 100 to 1000 colonies per plate and were plated on a medium to counter'select and/or differentiate male and female colonies. l5 Centrifuge Methodv-The cell suspensions were warmed for 10 minutes on a temperature block at 37 C and 4 ml of each were placed in prewarmed centrifuge tubes. The tubes were placed in a centrifuge equilibrated at 37 C and spun for a period of 5 minutes at 12,100 x g. The total run required 16 1/2 minutes; the raising of the powerstat from a setting of 0 to 50 required 105 seconds. The cells were agitated on a Vortex Junior for 2 minutes, diluted 10“, and placed in ice until usage. The remaining procedure followed as above (flask method). Millipore Method—-The cell suspensions were not prewarmed, but placed in ice until usage. One ml of the male and one ml of the female were placed upon a 0.45 uHA millipore filter (Millipore Filter Corp.) without a supporting pad and rotated gently for 60 seconds. Vacuum was then applied, impinging the bacteria to the millipore (Matney and Achenbach, 1962). The filters were then trans- ferred to prewarmed nutrient soft agar and placed at 37 C for the desired period of time (15, 30, 60, or 120 minutes). Zero time did not begin until the filters were on the agar. The samples were then either removed immediately, placed in a 50 m1 beaker containing 10 m1 of saline, and diluted 105 before placing in an ice bath, or transferred to a cold nutrient soft agar plate and placed in the refrigerator until usage. The remaining procedure followed as above (flask method). RESULTS Three methods were utilized to examine the fre- quency of transfer of the F—lac+ by conjugation. In each case frequency of transfer and host—controlled modification were specifically sought. In each series of crosses E. 33;; AB785 F-lac+ served as the initial donor of the genetic material. Thereafter, the infected recipients were used as donors in their respective crosses. Series I In the first series of crosses E. 33;; AB785 F-lac+ was mated with a homologous E. ggEEK-l2 recipient (AB113 lac'). E. _c_o_;i_ AB113 F-lac+ was then mated with E. 39;; AB266 lac’. These tranfers were initiated to fulfill the requirements governing host modification and restriction and also to serve as a control for subsequent experiments. The following graphs (Figures 2 and 3) indicate the rate of transfer of genetic material during a two hour period. It should be noted that the frequency of transfer with the millipore method was much greater than with either the centrifuge or flask method. In addition restriction occurred in the E. 33;; AB113 F-lac+ X E. 33;; AB266 lac’ cross as observed by the low frequency of transfer. Table IV lists the frequency of transfer and Figure 1 summarizes the indicated crosses. 16 17 S E. coli AB785 F-1ac+ str coli AB113 lac" strr ILTJ \/ + E. coli AB113 F-lac str coli AB266 lac- strr ‘\\\JE. coli AB266 F—lac+ str Figure l.--Summary of the transfer of F-lac+ in the control crosses of Series I. Percent Transfer 18 IOQ" 901 80“ 601 50+ .fl—O 4 i 0 //,/’ l””,// o X/ / / 30__ / 20“ 2X 10 / L l ‘ l I T I I I l 15 30 60 90 120 Time of Contact (Minutes) Figure 2.—-Frequency of transfer of F-lac+ from E.» coli AB785 F-lac+ to E; coli AB113 lac' via millI? pore (A), centrifuge (X), and flask (0) methods. Percent Transfer 19 1407- 1 3? 2Q_ 10- «Afr—fix —-—/ 0 f’ , 4 . . 15 30 6O 90 120 Time of Contact (Minutes) Figure 3.--Frequency of transfer of F—lac+ from E. coli A3113 F-1ac+ to E. coli AB266 lac" via milI‘i- pore (A), centrifuge TX), and flask (0) methods. 20 TABLE 4.--Frequency of transfer of F-lac+ with restricting and nonrestricting recipients.” After 60 Minutes CPOSS Method Recipient Recombinant Frequency 785 x 113 Millipore 2.3 x 108 2.1 x 108 9.1 x 10"1 Centrifuge 1.3 x 108 4.7 x 107 3.6 x 10-1 Flask 1.9 x 108 8.9 x 107 4.7 x 10'1 113 x 266 Millipore 2.0 x 108 3.4 x 107 1.7 x 10-1 Centrifuge 8.9 x 107 2.0 x 106 2.2 x 10‘2 Flask 2.7 x 108 8.0 x 106 3.0 x 10‘2 ”The donors were mixed with the recipients in a ratio of approximately 10 to 1 to give a total cell concentration of about 109 per ml. Variations in the ratio from 5-15 to 1 did not markedly affect the frequency of transfer. 21 Series II In the second series of crosses E. 32;; AB785 F-lac+ was mated with E. 93;; 33 lac". The initial mating indicated the presence of restriction when com- pared with the control matings of Series I. However, passage of the F—lac+ then proceeded quite readily from _E_. g_o_l_i_ 33 F-lac+ to E. _c_o_1_i_ 33 lac" as would be assumed by host modification. Subsequent passage into E. 33;; AB113 lac‘ also gave results that indicated little, if any, restriction. Again in these crosses, the milli— pore method produced better results than either the filter or centrifuge method (Figures 5, 6, and 7). During the production of streptomycin resistant mutants, two types of E. 32;; BB lac' strr mutants were detected. One mutant, designated as "high" received donor material with a frequency five times greater than another mutant labelled as "low" when employing the cen- trifuge method. All the experiments reported were per— formed with the "high" mutant. A contradiction to the above occurred when E. 32;; BB F-lac+ was mated with E, pullorum 35 laC' (Figure 8). An increase of transfer to 21% at 120 minutes occurred via the centrifuge method; however, as noted by the graph, a quick rise and then a sharp decline was observed when using the millipore method. Table V lists the frequency of transfer and Figure 4 summarizes the above crosses. 22 S E. coli BB lac- strr E. coli AB785 F-lac+ str S (str ) E. coli 33 F-lac+ str: (str ) E. coli BB lac- strr /_ \. coli BB F-lac+ strr Itxi E. coli AB113 lac‘ str{\\\\$ E. coli A3113 FL-lac+ strE// E. pullorum 35 lac- strr / ‘\$§3 pullorum 35 F-lac+ str 1" \/ Figure 4.--Summary of the transfer of F-lac+ in Series II. Percent Transfer 100 90 60-. 50 30.. 20.“. 23 Time of Contact (Minutes) Figure 5.--Frequency of transfer of F—lac+ from E. coli AB785 F-lac+ to E. coli BB lac- via millipore (a), centrifuge—(X), and flask (0) methods. Percent Transfer 21 led- 9Q- 80- 7Q- 6Q- 5Q. ”0t 3Q- 20‘ /JK""“'——""—_ 10- ///’ O A J L 4 J l I I I f 15 30 60 90 120 Time of Contact (Minutes) Figure 6.—-Frequency of transfer of F-lac+ from E. coli 33 F-lac+ to E. coli 33 laC“ via millipore ‘ (A), centrifuge (X), and flask (0) methods. Percent Transfer 100-- 90.. 80. 601. 50.. 40.— 30-- 20.- /o/ 25 / ,zr 1” / ”' l/X’I’I” 10.. // ‘//,//’ o<;//¥// 4*“ Ii /3 A A l I I 15 30 6O 90 120 , Time of Contact (Minutes) Figure 7.-—Frequency of transfer of F-lac+ from E. coli 33 F-lac+ to E. coli AB113 lac‘ via millipore (A), centrifuge (X), and flask (0) methods. Percent Transfer 26 I 3% 20. 10” Time of Contact (Minutes) Figure 8.—-Frequency of transfer of F-lac+ from E. coli BB F-lac+ to E. ullorum 35 lac- via.millip3re (A), centrifuge (I), and flask (0) methods. 27 TABLE 5.--Frequency of transfer of F-lac+ with restricting and nonrestricting recipients.* After 60 Minutes Cross Method Recipient Recombinant Frequency 785 x BB Millipore 3.4 x 108 1.1 x 108 3.2 x 10'"1 Centrifuge 1.5 x 108 1.0 x 107 6.7 x 10'2 Flask u.7 x 108 5.6 x 106 1.2 x 10‘2 33 x 33 Millipore 2.2 x 108 2.0 x 108 9.1 x 10‘1 Centrifuge 1.5 x 108 3.0 x 107 2.0 x 10‘1 Flask 1.5 x 108 1.1 x 108 7.3 x 10'1 33 x 113 Millipore 1.9 x 108 1.5 x 108 7.9 x 10‘1 Centrifuge 1.3 x 108 1.8 x 107 1.4 x 10"1 Flask 1.1 x 108 2.1 x 107 1.9 x 10'1 33 x SP Millipore 2.8 x 108 2.0 x 107 7.1 x 10'2 Centrifuge 1.u x 108 2.5 x 107 1.8 x 10‘1 Flask 1.4 x 108 6.3 x 106 u.5 x 10'2 *The donors were mixed with the recipients in a ratio of approximately 10 to 1 to give a total cell concentration of about 109 per m1. Variations in the ratio from 5-15 to 1 did not markedly affect the frequency of transfer. 28 Series III. In the third series of matings E. 23l£.AB785 F-lac+ was crossed with E. pullorum 35 lac“. Subsequent crosses were then made with E. pullorum 35 F-lac+. The millipore, centrifuge, and flask methods gave fairly equivalent results throughout this series of matings. The frequency of transfer with E. £2l$.AB785 F-lac+ was lower than that observed for any of the other initia1 crosses. The most significant difference, however, occurred when E. pullorum 35 F-lac+ was mated with either E. 33;; BB lac' or E. 32;; AB113 lac‘. Very little, if any, transfer was observed; in some experiments none was observed. When E. pullorum 35 F-lac+ was crossed with E. pullorum 35 lac’, the frequency of transfer increased to an observable rate, but still did not nearly approach the 100% level that would be expected with a homologous system. Table VI and Figurele and 11 give the percent transfer during the two hour interval, while Table VII lists the frequency of transfer at 60 minutes. Figure 9 gives a summary of the crosses. 29 E. coli AB785 F-lac+ strs E. pullorum 35 lac_ str: (str ) \/ E. pullorum 35 F-lac+ str: (str ) E. pullorum 35 lac- strr E. pullorum 35 F-lac+ strr E. coli A3113 lac- str{\\\\$ . coli AB113 F-lac+ sterK/ IN. I‘ [[11 . coli BB lac- str . coli BB F-lac+ strr [[211 Figure 9.-bSummary of transfer of F-lac+ in Series III. .-.i-u (I..~i~..n._. .-w..c...-av.~r|v.- Percent Transfer 100 90 5c- U0" 3G 20" lO-- 30 - o—————-——o——-———-.—-——-——o t 1 l 15 30 60 90 120 Time of Contact (Minutes) Figure lO.--Frequency of transfer of F-lac+ from E. coli AB785 F-lac+ to §. pullorum 35 lac‘ via millf; pore (A), centrifuge ( flask (0) methods. Percent Transfer 31 lO~ 1 15 30 60 90 120 Time of Contact (Minutes) Figure ll.-—Frequency of transfer of F-lac+ from S. ullorum 35 F-lac+ to E. ullorum 35 lac“ via milli- pore ZA), centrifuge (Xi, and flask (0) methods. 32 TABLE 6.--Frequency of transfer of F-lac+ from S. pullorum 35 F-lac+ to E. coli AB113 lac' and E. coli BB-lac-. _— w _L Method Cross Minutes Millipore Centrifuge Flask SP x 113 15 -——— __-_ ___- 3o ---- ---- ---- 60 .121 .171 .38% 120 .171 1.3% .uoz SP x BB 15 ——-- ___- _-__ 3o ---- ---- ---- 6O —--- ---- ---- 120 .05% .10% .05% 33 TABLE 7.--Frequency of transfer of F-lac+ with restricting and nonrestricting recipients.* After QQ-Minutes Cross Method Recipient Recombinant Frequency 785 x SP Millipore 2.1 x 108 2.5 x 107 1.2 x 10‘1 Centrifuge 8.7 x 107 1.6 x 107 1.8 x lo'1 Flask 1.2 x 108 1.7 x 106 1.u x 10‘2 SP x SP Millipore 7.8 x 107 6.0 x 106 7.7 x 10-2 Centrifuge fl.3 x 107 1.7 x 106 h.0 x 10-2 Flask 5.2 x 107 1.0 x 106 1.9 x 10‘2 SP x 113 Millipore 5.u x 108 6.6 x 105 1.2 x 10'3 Centrifuge 1.9 x 108 3.3 x 105 1.7 x 10‘3 Flask 3.u x 108 1.u x lo6 u.l x 10‘3 SP x BB Millipore 7.5 x 108 3.3 x 105 u.u x 10’“ +Centrifuge 3.“ x 108 3.3 x 105 9.7 x 10‘“ fFlask 6.8 x 108 3.3 x 105 H.9 x 10-“ *The donors were mixed with the recipients in a ratio of approximately 10 to l to give a total cell concentration of about 109 per ml. +After 120 minutes. Variations in the ratio from 5-15 to 1 did not markedly affect the frequency of transfer. DISSCUSSION Methods of Conjugation Three series of crosses were initiated to determine the frequency of transfer by the millipore, centrifuge, and flask methods. In all crosses but one (E. 39;; AB785 F—lac+ X E. pullorum 35 lac'), the millipore method was found to be the most efficient means of transferring the F-lac+ from donor to recipient strains. In many of the crosses, the difference in rate of transfer between this method and the other two was great enough to preclude any error that might have been incurred in technique. An explanation for the millipore method giving the best results might be related to the following. The bacteria are impinged upon a solid surface in proximity to one another and thus the rapid separation encountered in broth is eliminated (Matney and Achenbach, 1962). However, they are still in proximity with the atmosphere and can continue receiving a constant supply of nutrients-- both factors which appear to be necessary for conjugating cells (Fisher, l957). The above results were inferred from platings in which the number of bacteria had increased, sometimes to such an extent that the usual dilution was not sufficient for the reading of results. 34 35 The centrifuge method produced results that were usually comparable to those obtained by the flask method (exceptions: Figures 6, 8, and 10). In these crosses the bacteria did not readily multiply while in the pellet; this suggests that Optimal conditions for growth were not present. The exception noted in Figure 6 occurred when E. 22;; BB F—lac+ was crossed with E. 92;; BB lac“. A possible explanation might be gleaned from the clumping that occurred when the cells were suspended. Perhaps the clump- ing between these two strains of E. 32;; BB had the same physical effect as that encountered with the millipore method. The two other exceptions will be discussed later. Utilizing the flask method, a rapid multiplication of the bacteria occurred with time. However, as noted by a few of the graphs, the percent transferred did not-rise considerably with this method when compared with the cen- trifuge method. Whenever an E. ggEEhK—l2 or BB was mated with a E. pullorum, the results tended to improve if the centrifuge method were employed. This suggests that the packing of cells gradually permits a greater percentage of transfer than could otherwise be obtained by the millipore method. Perhaps either the proximity or the configuration of the cells plays a role. Therefore, by experimenting with all three methods, it appears that many of the results in the literature could 36 be improved upon by using that method that most readily facilitates the transfer of the genetic material whether it be of a Hfr, F', or F+ variety. Host-controlledfiModification The presence of host-controlled modification was found in E. pullorum 35. The F-lac+ was transferred from E. 22;; AB785 F—lac+ to E. pullorum 35 and then passed to another E. pullorum 35 at a reasonable, but low rate. Restriction probably plays a role in both the initial and homologous cross; nonhomology should be nonexistent since integration does not necessarily occur (Arber and Morse, 1965).' Even though restriction appears to be present in both the initial and homologous cross, it may be coupled with effective contact. The decrease in the efficiency of transfer between an E. coli—E.pullorum or E. pullorum- E. pullorum cross when compared to an E. ggEEfE. 33;; cross suggests that a missing surface component may contribute to this decrease. Mutants have been isolated which have an increased ability to act as recipients; neither tech— nique nor condition has been discovered which impairs the ability of the cell to act as recipient (Gross, 196”). The possibility exists that the F-lac+ might carry the host specificity from its previous host. However, by repeated isolations, any host specificity should have been lost during replication. When the E. gullorum was used as the donor to infect E. coli AB113 or BB, the frequency of 37 transfer dropped significantly which would imply that the F-lac+ in the E. pullorum had been modified. Another interesting result was observed in the matings of the control crosses. Although no restriction would be expected in a homologous cross between various strains of E. ggEE_K-12, some was noted. This restriction also has been noted by Makela gE_§E.(l962) and Arber and Morse (1965). The crosses of Series II also yielded some note- worthy information. Whereas some restriction was noted in the initial cross (3. 911.4. AB785 F-lac+ x g. _c_o_l_i_ BB lac’), none was noted when E. 32EE_BB F-lac+ was crossed with a homologous recipient or with E. 39;; AB113 lac'. Therefore, it appears that both recipients are capable of receiving a modified F—lac+ equally well and that the mechanism involved in host-controlled modification may not be as selective as might be hypothesized or that E. £2l$ K-12 and BB are more homologous than assumed. Perhaps the base sequence is modified by methylation (Arber, 1965; Klein and Sauerbier, 1965) so that it is recognized by both the nucleases present in E. ggEE.K—l2 and BB as "self," rather than "nonself." When E. 2222.33 F-lac+ from Series II was crossed with E. pullorum-35, a gradual rise in percent transfer occurred. However, when the same cross was performed via the millipore method, a rapid increase, followed by a gradual decline 38 was observed. Arber and Morse (1965) observed similar results when they crossed F+ K-l2 gal“ X F“ K-l2(Pl) gal+ and F+ K-l2 gal’ x F- B gal+. They suggested that the lethality was only present in those cell pairs that persisted after the end of donation of the transferred DNA molecule. Clowes (1963) and Gross (1963) both ob— served that lethality occurred at a high ratio of Hfr to F' cells and surmised that these effects were due to damage to F‘ cells. Gross also found that not all of his Hfr strains produced this effect. Thus, perhaps in this cross, the conditions imposed by the millipore filter, i.e., the immediate proximity of the bacteria to one an- ‘ other, also cause this lethality. SUMMARY Of three methods utilized in the transfer of the intermediate sex factor, F-lac+, the millipore method was found to be generally superior to either the centrifuge or flask method. The transfer of F-lac+ also was affected by the strains involved. E. 32;; AB785 was used as the universal donor of F-lac+. Fulac+ was transferred readily to both an E. 33;; K—12 and BB, but with a lower frequency to a E. pullorum. Subsequent transfer with E. ggEE_F-lac+ to both E. ggEE| BB lac“ and AB113 lac‘ readily occurred, but was method- dependent when E. pullorum 35 was used as the recipient. When E. pullorum 35 was used as the donor, restric- tion was readily observed if E. 32;; A3113 or BB were the recipient. When E. pullorum 35 was the recipient, some restriction was noted. 39 BIBLIOGRAPHY Adelberg, E. A. and S. N. Burns. 1959. A variant sex factor in Escherichia coli. Genetics 53:497. Adelberg, E. A. and S. N. Burns. 1960. Genetic varia- tion in the sex factor of Escherichia coli. J. Bacteriol. 12:321-330. Arber, W. 1965. Host specificity of DNA produced by Escherichia coli. V. The role of methionine in the production of host specificity. J. Mol. Biol. EE:2h7-256. Arber, W. and D. Dussoix. 1962. Host specificity of DNA produced by Escherichia coli. I. Host controlled mgdigication of bacteriophage A. J. Mol. Biol. E: 1‘3 0 Arber, W. and M. L. Morse. 1965. Host specificity of DNA produced by Escherichia coli. VI. Effects on bacterial conjugation. Genetics Elle7-1A8. Bertani, G. and J. J. Weigle. 1953. Host controlled variation in bacterial viruses. J. Bacteriol. EEzll3-l2l. Boyer, H. 1964. Genetic control of restriction and modification in Escherichia coli. J. Bacteriol. §_8_:l652-l660 ‘— Clark, A. J. and E. A. Adelberg. 1962. Bacterial conjugation. Ann. Review Microbiol. Egz289-3l9. CloWes, R. C. 1963. Colicin factors and episomes. Genet. Res. 3:162—166. Colson, C., S. W. Glover, N. Symonds, and K. A. Stacey. 1965. The location of the genes for host-controlled modification and restriction in Escherichia coli K—l2. Genetics EE:10A3-1050. Echols, H. 1963. Properties of F' strains of Escherichia coli superinfected with F-lactose and F-galactose episomes. J. Bacteriol. EE:262+268. 40 41 Fisher, K. W. 1957. The role of the Krebs Cycle in con- jugation in Escherichia coli K-12. J. Gen. Micro— bio. 16: 120- 135. Glover, S. W., J. Sche11,N. Symonds, and K. A. Stacey. 1963. The control of host- induced modification by phage P1. Genet. Res. 3:480—482. Gold, M. and J. Hurwitz. 1964a. The enzymatic methyla- tion of ribonucleic acid and deoxyribonucleic acid. V. Purification and properties of the deoxyribonucleic acid-methylating activity of Escherichia coli. J. Biol. Chem. 239: 3858- 3865. Gold, M. and J. Hurwitz. 1964b. The enzymatic methyla— tion of ribonucleic acid and deoxyribonucleic acid. VI. Further studies on the properties of the deoxyribonucleic acid methylation reaction. J. Biol. Chem. Eigz3866-3874. Gross, J. D. 1963. Cellular damage associated with multiple mating in E. coli. Genet. Res. 3:462—469. Gross, J. D. 1964. Conjugation in bacteria, p. 1—48. In I. C. Gunsalus and R. Y. Stanier [ed. ], The Bacteria. Volume V: Heredity. Academic Press, Inc., New York. transfer and multiplication of sexduced cells. Genet. Res. 3:30—41. Hayes, W. 1953a. Observations on a transmissible agent determining sexual differentiation in Bacterium coli. J. Gen. Microbiol. Ez72-88. Hayes, W. 1953b. The mechanism of genetic recombination in E. coli. Cold Spr. Harb.Symp. Quant. Biol. l_§_:75-93. Hayes, W. 1964. The genetics of bacteria and their viruses. John Wiley and Sons, Inc., New York. Hirota, Y. 1960. The effect of acridine dyes on mating type factors in Escherichia coli. Proc. Natl. Acad. Sci. U. S. 46: 57—64. Hoekstra, w. P. M. and P. G. de Haan.'”1965. The location of the restriction locus for A. K in Escherichia coli- B. Mutation Res. 2. 204-212. 42 Jacob, F. and E. L. Wollman. 1958. Genetic and physical determinations of chromosomal segments in E. coli. Symp. Soc. Exptl. Biol. EE:75-92. Jacob, F. and E. L. Wollman. 1961. Sexuality and the genetics of bacteria. Academic Press, Inc., New York. Klein, A. and W. Sauerbier. 1965. Role of methylation in host controlled modification of phage T1. Biochem. Biophys. Res. Commun. EE:440-445. Lederberg, J. 1948. Detection of fermentative variants with tetrazolium. J. Bacteriol. E§3695. Lederberg, J. 1950. Isolation and characterization of biochemical mutants of bacteria. Methods Med. Res. E:5—22. Lederberg, J. and E. L. Tatum. 1946a. Novel genotypes in mixed cultures of biochemical mutants of bacteria. Cold Spr. Harb. Symp. Quant. Biol. EE:113-114. Lederberg, J. and E. L. Tatum. 1946b. Gene recombination in E. coli. Nature 158:558. Lederberg, S. 1957. Supression of the multiplication of heterologous bacteriophages in lysogenic bacteria. Virology 3:496-513. Ledinko, N. 1964. Occurrence of 5-methy1deoxycytidylate in the DNA of phage lambda. J. Mol. Biol. E:834-835. Luria, S. E. and M. L. Human. 1952. A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol. Efl:557-569. Makela, P. H., J. Lederberg, and E. M. Lederberg. 1962. Patterns of sexual recombination in enteric bacteria. Genetics 31:1427-1439. Matney, T. S. and N. E. Achenbach. 1962. New uses for membrane filters. III. Bacterial mating procedures. J. Bacteriol. Efl:874—875. Pittard, J. 1964. Effect of phage-controlled restriction on genetic linkage in bacterial crosses. J. Bacteriol. El:l256-1257. Pittard, J. and E. A. Adelberg. 1964. Gene transfer by F' strains of Escherichia coli K-12. III. An analysis of the recombination events occurring in the F' male and in the zygotes. Genetics 32:995-1007. 43 Stacey, K. A. 1965. Intracellular modification of nucleic acids. Brit. Med. Bull. EE:211—216. Watanabe, T. and T. Fukasawa. 1961. Episome—mediated trans— fer of drug resistance in Egterobacteriaceae. II. Elimination of resistance factors withwacridine dyes. J. Bacteriol. EE:679-683.