A CONJUGATION SYSTEM IN SALMONELLA PULLGRUM Tuluesis for ”18 Degree of pin. D. MICHIGAN STATE UNIVERSITY Mary Judith Robinson 1964 1145515 This is to certify that the thesis entitled A Conjugation System in §§jmoneila pullorum presented by Mary Judith Robinson has been accepted towards fulfillment of the requirements for Ph.D. degree in Microbiology 5 Pub] IC Health // //;z;¢L}¥ fiéilaxL.y Mk\§ Delbert E. Schoenhard Major professor Date September 3, 1961+ 0-169 LIBRARY Michigan State University ABSTRACT A CONJUGATION SYSTEM IN SALMONELLA PULLORUM by Mary Judith Robinson A conjugation system was established in Salmonella pullorum by the identification of a naturally occurring high frequency recombi- nation (Hfr) strain, §. pullorum 6, and by the introduction of F+ and F'lac+ sex factors into g. pullorum 35 followed by the isolation of Hfr mutants of these cultures. The maleness of F+, F', and Hfr strains of §. pullorum was determined by a positive staining reaction, ability to transfer the staining reaction, ability to be cured of the staining reaction, ability to conduct chromosomal markers to recipient strains, and insusceptibility to a female specific _S_. pullorum bacterio- phage. §. pullorum 35 was shown both cytologically and genetically to be a recipient population. Electron microscopic examination of mating mixtures demonstrated the ability of §. pullorum 35 to form effective conjugal pairs with and to receive genetic material from an E. coli donor. Genetic evidence indicated that strain 35 is an F- rather than F0 recipient. Two Hfr strains of §. pullorum with different origins and opposite orders of entry of genetic characters were used to determine the positions of several markers on the donor chromosome. Three mapping procedures--gradient of transmission, genetic constitution of recom— binants, and interrupted mating- -were employed. The order of markers on the §_. pullorum donor linkage group was found to be his--(pro)-- 1eu—-(ara)--mot--cys--(ile)--mtl--ga1--str--xyl. The §. pullorum l Mary Judith Robinson chromosome was shown to be a closed continuous structure which becomes discontinuous and linear during transfer from an Hfr strain. _S_. pullorum Hfr-1 behaved classically with regard to the con- duction of genetic determinants to recipient cells. Both Hfr-l and §_. pullorurn 6 formed unusually stable effective pairs with an _S_. pullorum recipient. A CONJUGATION SYSTEM IN SALMONELLA PULLORUM By Mary Judith Robinson 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 1964 This thesis is dedicated to my parents who gave me an intellect and to the educators who developed it. ii ACKNOW LEDGEMENTS I wish to express my sincere appreciation to Dr. Delbert E. Schoenhard for his patience, encouragement, and guidance throughout the course of this study and especially for his continued interest and the constant challenge his suggestions presented. I should also like to acknowledge the aid and constructive criticism of my fellow graduate students. A special note of thanks is due to Mr. S. S. Tevethia for his valuable advice and assistance. During the course of this study, I was supported in part by a National Science Foundation Summer Fellowship for Graduate Teach- ing Assistants. M.J.R. iii TABLE OF CONTENTS Page INTRODUCTION ........................ 1 LITERATURE REVIEW .................... 3 Review of the Conjugation Process ............ 3 Mating types of bacteria ............... 3 Alteration of mating type ............... 4 Classification of fertility factors. . . ...... . . 4 Modification of fertility factors ......... 6 Successive steps in the conjugation process ..... 7 Frequency of genetic transfer during conjugation . . 9 Genetic Analysis of a Linkage Group by Conjugation . . . 11 Mapping by the gradient of transmission ....... 11 Mapping by genetic analysis of recombinants . . . . 12 Mapping in time units. ................ 12 Pictorial Representation of the E. (5111 and _S_. t himur- i_u_r_n_ Chromosomes .................. 12 Occurrence of Intra- and Intergeneric Conjugation. . . . 12 MATERIALS AND METHODS Cultures . . ............ . . . ......... 17 Media . . ......................... 17 Composition of synthetic media ........... 1? Composition of complex media ............ 21 Reagents . ..... . ................... 25 Mutation Procedures ............... . . . . 26 Induction of mutations with 2-aminopurine ...... 26 Isolation and selection of mutants .......... 26 Isolation of fermentation mutants ........ 26 Selection of amino acid and nucleotide mutants by the penicillin method . . . . . ._ .. .. . . 26 Selection of amino acid and nucleotide mutants . 28 by thymineless death . . . , , . _ . . . . . 28 Detection of Male Cultures . . ., ........... . . 28 Altered surface properties . . . . . ......... 28 The staining reaction .............. 28 iv TABLE OF CONTENTS - Continued Page Insusceptibility to a female specific .pullorum bacteriOphage ...... . . . 30 Transfer— Sof sex factors (tube mating method): . . 30 Transfer of chromosomal markers in F x F crosses ..................... 3O Curing of; F and F' cultures with ac ridine dyes . . 31 Preparation of Mating Mixtures for Electron Mic ro- sc0pic Examination ......... . . ...... 32 Negative staining with phosphotungstic acid ..... 32 Positive staining with uranyl acetate ....... . 32 Shadow casting .................... 34 Isolation of High Frequency Recombination (Hfr) Strains 34 Mating Procedures for Hfr x F- Crosses ........ 35 Gradient of transmission . ............. 35 Mapping in time units . . . .......... . . . 35 Syringe method bn interruption ......... 35 Waring blendor method of interruption ..... 36 Genetic analysis of recombinants . . . . ...... 36 RESULTS Induction of Mutation with 2- -Aminopurine and Selection of Mutants ................. . . . 37 Survey of§. pullorum Bacteriophages for Male or Female Specificity .................. 37 Survey of _S_. pullorum Strains for Maleness ..... . . 37 Transfer of Sex factors to_ S. pullorum .......... 41 Introduction of an F se-Bt factor to S. pullorum 35W 41 Introduction of an F'lac episome into S. pullorum. 42 Evidence of Donor Ability in S. pullorum 35F+ . 44 Transfer of a+sex factor by _S_. pullorum 35F+W6. . . 44 Removal of F sex factors with ac ridine dyes. . . . 45 Low frequency transfer of chromosomal markers by_§. pullorum 35F+W6 ......... . . . . 47 Evidence of Donor Ability in g. pullorum 6 . . . . . . . 53 Evidence of Donor Ability in S. pullorum strr F'lac strains ........... ———_ . . . . . . . 54 Transfer of an F'lac+ particle by _S_. pullorum 355trrF'lac+ cultures ....... . . . . 54 TABLE OF CONTENTS - Continued Efficiency of transfer of an F'lac+ plasmid from E. pullorum 35F'lac+ to E_. pullorum 35strr .................... Removal of F'lac+ factors with ac ridine dyes. . . . Transfer of chromosomal markers by strr F'lac+ cultures of E. pullorum ............. Evidence of a Conjugation Process ............ Electron microscopic evidence of conjugation be- tween E. coli AB785 and E. pullorum 355tr1'. . A. Formation of effective conjugal pairs. . B. Transfer of genetic material ........ Genetic evidence of cell contact ........... Isolation of Hfr Strains from F+ and F' E. pullorum Cultures ....................... Recipient Ability of _S_. pullorum 35 ........... Evidence of Intrastrain Conjugation ........... Conjugational Analysis of the _S_. pullorum Chromosome Analysis of the _S_. pullorum linkage group by inter- generic conjugation ............... Kinetics of effective contact between E. pul- lorum and E. coli AB113 .......... Location of markers on the distal segment of the E. pullorum Hfr-l linkage group. . . . Conjugational analysis of the E. pullorum chromo- some by intraspecies conjugation . . . . An intrastrain conjugation with E. pullorum Hfr-1 . . . . ...... . ......... An interstrain conjugation with E. pullorum 6 . DISCUSSION ........... SUMMAR Y ........................... BIBLIOGRAPH Y ........................ vi Page 55 57 58 62 62 62 67 73 78 78 79 80 80 88 88 93 93 98 106 107 LIST OF TABLES TABLE Page I. Characteristics of bacterial strains. ....... . 18 II. A list of media used and the purpose of each . . . . 19 III. Composition of amino acid and nucleotide pools. . . 22 IV. Supplements added to E or W medium for specific selections ............. . . . . ..... . 23 V. Concentrations of acridine dyes used for curing F+ and F' cultures. . . .......... . .1. . . . . 33 VI. Types of mutants. isolated following 2-aminopurine induction .............. . . . . . . . . . 38 VII. Survey of _S_. pullorum bacteriophages for sex specificity . . . . . . . .............. . 40 + VIII. Curing of F strains with acridine orange . . . . . 46 IX. Curing of E. pullorum 6 with low concentrations of acridine orange. . . . . . . . . . . . . ....... 46 + X. Curing of F strains with neutral red . ....... 48 XI. Transfer of chromosomal markers by positively staining and cured cultures ............ . 52 + XII. Curing of F'lac E. pullorum strains ........ 59 XIII. Kinetics of union formation between E. pullorum Hfr-l and E. coli ABll3 ........ . . . . . . . 89 . . . + + XIV. Genet1c const1tut1on of gal strr and mtl strr recombinants ..................... 92 vii LIST OF TABLES - Continued TABLE Page XV. Transmission of markers from E. pullorum Hfr-l to E. pullorum 358trrhis‘pr0'ile'ara'mtl-gal". . . 95 XVI. Genetic constitution of recombinants in an Hfr-1 x E. pullorum 35strrhis“pro-ile'ara‘mtl'gal" cross. 95 XVII. Transmission of markers from E. pullorum 6 to E. pullorum 353trrhis'ile'ara'mtl'gal‘xyl" . . . . 99 XVIII. Genetic constitution of recombinants in an E. pullorum 6 x _S_. pullorum 353trrhis'ile'ara’mtl' gal'xyl" cross ...... . ..... . . . . . . . . 99 viii LIST OF FIGURES FIGURE Page 1. Positions of some markers on the circular E. coli linkage group ..................... 13 2. Positions of several markers on the circular E. typhimurium linkage group .......... . . 14 3. Regions of homology between the E. coli and E. £yphimurium chromosomes. . ......... . 15 4. Selection of mutants by thymineless death in E. pullorum 35 . ................ . . . 39 5. Transfer of an F+ sex factor to E. pullorum 35W. . 43 6. Transfer and curing of the staining reaction . . . . 49 7. Transfer of chromosomal markers from S_. pullorum 35F+W6 to E.<_:_o_1_1104. . . . ............ 51 . . + 8. Eff1c1ency of transfer of the F'lac plasmid from E. pullorum 35F'lac+ to E. pullorum 353trr ..... 56 + 9. Transfer and curing of F'lac particles in E. pullorum 35 .................... 6O 10. Contact between an E. pullorum and an E. coli cell. 63 11. Disappearance of cell boundaries between members of an E. coli-_S_. pullorum conjugal pair . ...... 65 12. Demohstration of a cyt0plasmic bridge connecting members of; an E. coli-E. pullorum conjugal pair . 66 13. Overlapping cells of E. pullorum and E. coli stained with uranyl acetate . . ..... . . . . . . 68 ix LIST OF FIGURES - Continued FIGURE 14. 15. l6. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Uranyl acetate stained E. pullorum . Transfer of DNA between members of an E. coli- E. pullorum conjugal pair . . . . . . . ..... Transfer of DNA between members of an E. coli- E. pullorum conjugal pair ............ E. coli cells connected by a DNA filled tube Plasmodesma connecting E. coli cells in a terminal stage of division .................. An E. coli cell in an early stage of division E. coli conjugal pair stained with uranyl acetate . An E. coli mating pair stained with uranyl acetate . Positions of the mot, cys, and leu genes on the E. pullorum Hfr-l linkage group as determined by inte rgene ric conjugation ............. Mapping of the proximal segment of the E. pullorum Hfr-l linkage group in time units .......... Relationship between percent male marker transfer and time of entry of male markers ......... Time of entrance of the his+ marker into E. coli AB113. . ....................... Growth curves of donor and recipient in an E. pullorum Hfr-l x E. coli AB113 mating mixture Kinetics of union formation between E. pullorum Hfr-1 and E. coli AB113 ........... , . . . Page 69 70 71 72 74 75 76 77 82 83 85 86 87 9O LIST OF FIGURES — Continued FIGURE Page 28. The order of several genes on the S_. pullorum Hfr-l linkage group ................. 94 29. The probable order of entry of markers on the E. pullorum 6 linkage group ............. 100 30. A composite picture of the circular E. pullorum linkage group .................... 105 xi INTRODUCTION The discovery of bacterial conjugation in Escherichia coli K-12 was perhaps the most important event in the recent history of bacterial genetics. The importance of this discovery lies in the fact that conju- gation permits a rapid genetic analysis of the entire bacterial chromo- some. Genetic analyses by conjugation have led to the pictorial repre- sentation of the bacterial chromosome as a circle each point of which represents a single mutation. The interval between two points signifies a quantitative expression of the amount of DNA separating two mutant sites. The existence of a circular linkage group of linearly-ordered genetic determinants is well established in E. c_ql_i_ and may be true of bacteria in general. Genes responsible for the synthesis of cellular metabolites and genes responsible for the utilization of exogenous energy sources have been shown to be arranged on the bacterial chromosome in two fashions. Most commonly genes involved in a specific pathway are located within a limited region of the chromosome in a coterminus arrangement. For several biosynthetic pathways a linkage of bio- chemically related genes is not observed. The eight genes controlling the formation of enzymes required for arginine synthesis, for instance, Occur in one cluster of four genes with the remaining four genes dis- tributed singly in widely separated areas of the E. <_:_o_li_ and Salmonella Eyphirnurium chromosomes. Evidence has accumulated which suggests that a correlation exists between virulence and the ability to synthesize arginine in Salmonella Bullorum. Because of the scattering of arginine loci on the bacterial chromosome and because bacterial conjugation permits a rapid gross structure analysis of the entire chromosome, conjugational analysis of the E. pullorum linkage group represents a rational approach to the future genetic study of virulence in this organism. The following study was undertaken to establish a conjugation system in E. pullorum and to genetically analyze the E. pullorum linkage group. This purpose was to be accomplished by the identifi- cation of a natural conjugation system or by the introduction of sex factors and subsequent isolation of mating types followed by recombi- national studies on the E. pullorum chromosome. LITERATURE REVIEW Review of the Conjugation Process Bacterial conjugation is most accurately and simply described as a unidirectional transfer of genetic material from one bacterial cell to another where the transfer requires contact between cells (Clark and Adelberg, 1962). Mating Types of Bacteria--Cultures of Escherichia coli capable of conjugating, or mating, fall into two classes: those which donate genetic material and those which receive genetic material. By anology to higher organisms the donor cultures are called male and the recipient cultures are called female. Male cultures owe their ability to donate to genetic elements known alternately as sex factors, fertility factors, or F-factors and are thus referred to as F+(Jacob and Wollman, 1961). Female cultures which lack fertility factors but which possess chromosomal markers enabling them to mate with male cells (Johnson, e_t a_._l. , 1964) are called F-. Four criteria distinguish a male from a female culture. (1) The surface properties of donor cells are altered so as to allow male bacteria to pair specifically with F- recipients (Jacob and Wollman, 1961). The difference in surface structure between donor and recipient bacteria is revealed by: (a) the ability of a male cell to form effective pairs with female cells, (b) the altered antigenicity of a. donor cell (Le Minor and Le Minor, 1956; Orskov, I. and F. Orskov, 1960), (c) the sensitivity of male cultures to male specific RNA con- taining bacteriophages (Loeb, 1960; Loeb and Zinder, 1961) and Conversely their resistance to female specific bacteriophages, (d) the permeability of a male cell to the dye eosin (Zinder, 1960b), (2) Donor cultures transfer promotor genes to recipient cells (Jacob and Wollman, 1961). (3) Donor cultures conduct chromosomal markers to recipient cells (Hayes, 1964). (4) All male characteristics may be eliminated from a donor population by treatment with ac ridine dyes (curing) (Hirota, 1960). o In Salmonella typhimurium a third class, the neuter or F class, has been described (Baron, e_t a}. , 1959). This class of bacteria is capable of neither donating nor receiving genetic information by means of conjugation. Alteration of MatinLType--The mating type of a cell may be altered in several ways. A male cell may be converted into a female cell by loss of the F-factor either as a result of spontaneous mutation (Jacob e_:_t a_l. , 1960) or experimental removal, resulting from treatment with acridine dyes (Hirota, 1960). An F0 cell may become F+ by spon- taneous mutation (Baron it aEI. , 1959). An F- cell may be converted to + an F type only by receiving an F-factor from a male culture (Jacob and Wollman, 1961). Classification of Fertility Factors--Fertility factors may be divided into two classes: (1) F-factors containing promotor genes only and (2) F-factors containing promotor genes plus one or more chromo- Somal genes attached to and transferred simultaneously with the pro- motor. Promotor genes are those genetic determinants responsible for the formation of effective conjugal pairs by alteration of the male cell surface, mobilization of the donor genetic material, and provision of an immediate energy source required for genetic transfer (Clark and Adelberg, 1962). The best known sex factor of the first type is the F-agent of £3. 21; K-12 (Cavalli c_t .11., 1953). An example of thesecond class of sex factors is the F' or sexducing (also called F-ducing or F-mero- genote) of E. c_oli (Jacob e_t z_1_l. , 1960). The size of the chromosomal segment which can be incorporated into a sex factor is variable. It is, however, generally small, consisting of only those genes associated with a single operon (Jacob and Wollman, 1961). The chormosomal region of an F-particle may, on the other hand, represent as much as one-tenth of the bacterial linkage group and may include genes responsible for as many as four separate biosynthetic pathways in E. 9521 (Pittard and Adelberg, 1964). . Each class of F-elements is divided into two subgroups: (a) episomes and (b) plasmids. Episomes are genetic elements capable of existing autonomously in the cytoplasm where they replicate inde- pendently of and more rapidly than the bacterial chromosome (Lederberg e_t a;1., 1952; Cavalli et 11., 1953) and of becoming integrated into the bacterial linkage group (Jacob and Wollman, 1961). When integrated the F-factor replicates in synchrony with the bacterial genome. In the auto- nomous state 'F-episomes are donated independently of chromosomal transfer to recipient cells at a high frequency. When an F-episome is integrated into the bacterial chromosome the entire chromosome may be transferred to recipient cells during conjugation. A mutant of an F+ culture containing integrated promotor genes is referred to as a high frequency recombination (Hfr) mutant, since, once isolated, this mutant culture will transfer chromosomal markers to recipient cells at a high frequency. An F-episome may become stably attached to the chromosome at any site. At whatever point the F-particle interacts with the chromo- _ Some the linkage group becomes linear (Clark and Adelberg, 1962). The linear chromosome is then transferable to recipient cells in an Oriented and sequential manner such that the point of interaction, the origin, enters first followed sequentially by chromosomal markers with the integrated sex factor (Hfr gene) entering last (Jacob and W011- man, 1961). An F' episome differs from an F-episome in its ability to inte- grate with the chromosome. The F' element is obligated to enter the chromosome at the site of whatever chromosomal gene it carries (Adelberg and Burns, '1959). The obligatory point of entrance is pre- sumably the result of a necessity of base pairing homology between the chromosomal marker episome and the chromosome. The result of this directed integration site is an Hfr strain with a predictable order of entry according to which the attached exogenotic marker enters late followed only by the terminal Hfr gene. Three criteria distinguish an Hfr from an F+ donor. (.1) An Hfr strain transfers genetic markers at a frequency at least one thousand fold greater than the F+ culture from which it was derived. (2) The transfer of markers occurs in an oriented manner such that every recipient cell mating with an Hfr donor receives the donor genetic characters in the same order. The transfer of markers in an F+ cul- ture, on the other hand, is random as a consequence of the presence of several Hfr mutants with differing orders of entry in the culture. (3) The promotor is donated at a low frequency in Hfr cultures rather than at the high frequency characteristic of F+ cultures. Plasmids, unlike episomes, are capable of existing solely in the autonomous state in the bacterial cytoplasm (Lederberg, 1952). Plasmids are donated to recipient cells at a high frequency, usually in the absence of chromosomal transfer (Clark and Adelberg, 1962). Modification of Fertility Factors--Fertility factors may be l"IIOdified in three ways; (1) by mutational loss of a promotor function; (2) by recombination with the'bacterial chromosome; or (3) by alteration of the physiological state of the donor. Ultraviolet treatment of strains of E. g}. possessing fertility factors produces cells which are male in all respects with the exception that they are no longer capable of transferring genetic material to recipient cells (Jacob and Wollman, 1961). Sex factors of this type have lost at least one promotor function and are referred to as mutant promotors. Cells containing modified F-particles are, in addition, in— capable of receiving genetic material from known donor strains (super- infection) by means of conjugation (Méikel'ai it 31., 1962). Such a mutant promotor may be responsible for the F0 nature of E. typhimurium cul- tures (Clark and Adelberg, 1962). During growth of Lac-/Lac+ F' heterogenotes, cells which exhibit the lac— phenotype of the parent recipient segregate. The genotypes of such segregants may be determined by genetic tests. A minority are haploid parentals. The majority, however, still carry an F'lac factor which, as a result of mitotic recombination carries the chromosomal lac- mutation (Jacob and Wollman, 1961). In such cases it is the attached chromosomal segment rather than the promotor itself which is altered. The physiological condition of the donor cell may also prevent expression of a promotor function. When mated in the stationary phase of growth, aerated, or when treated with periodate all cells in a male population phenoc0py F“ (cf. Clark and Adelberg, 1962). The original male phenotype is restored when the restrictive physiological conditions are removed and the cells are grown through one or two generations (Hayes, 1964) . Successive Steps in the Conj_ugation Process--The conjugation Process consists of four distinct steps: (1) formation of effective conjugal pairs; (2) conduction or donation of genetic material from the dOnor to the recipient cell; (3) expression of newly received genes; (4) formation of a stable recombinant cell. The formation of a conjugal pair procedes in three stages: (1) cells of opposite mating type collide and undergo an unstable attach- ment (contact); (2) the cell boundaries separating the two cells disappear in the area of contact; (3) a stable cytoplasmic bridge connecting the cells is formed (Jacob and Wollman, 1961). Contact between cells in a mating mixture is a random process depending solely upon chance collision. No attracting substance is pro- duced by either donor or recipient cells (Jacob and Wollman, 1961). Thus, donor cells may collide with other donor cells as well as with recipient cells. Two coupled cells of opposite mating type are referred to as a specific pair (Clark and Adelberg, 1962). A specific pair is con- verted into an effective conjugal pair when all the conditions necessary for genetic transfer--i. e. , formation of a cytoplasmic bridge, prepara- tion of the genetic material for transfer (mobilization), and provision of an immediate energy source for the process of transfer--have been achieved (Clark and Adelberg, 1962). Once the appropriate conditions have been established, donor genetic material is transferred to recipient cells. Two types of transfer are distinguished. In one case the promotor is transferred independently of the chromosome. In the second case the promotor and chromosomal markers are transferred as linked elements. The term conduction has been envoked to describe this second type of transfer (Clark and Adelberg, 1962). Transfer of genetic material is followed by expression of the newly received DNA immediately (Riley e_t aEI. , 1960) or within several generations. The time required for expression depends upon the com- plexity of the introduced character (Jacob and Wollman, 1961). The events which occur between the time of expression of trans- ferred genes and the formation of a stable recombinant cell are unknown. Conjugational analyses have been of little help in the elucidation of the mechanism of recombinant formation in exconjugate cells (Clark and Adelberg, 1962) . Frequency of Genetic Transfer during Conjugation--The frequency with which chromosomal markers are transferred to recipient cells during conjugation depends upon (1) the location of the F-factor in the donor cell and (2) the recipient capacity of the female culture. A Male cultures with F-episomes primarily in the autonomous state transfer chromosomal markers to F' cells at a low frequency as measured by the number of recombinant cells formed. The frequency of recombinants formed in low frequency recombination crosses (F+ x F') does not exceed one recombinant cell per 105 donor cells (Jacob and Wollman, 1961). The formation of these recombinants is due to the occurrence of rare Hfr mutants in the F" population. Male cultures containing integrated sex factors (Hfr strains) transfer chromosomal markers to F- cells at a high frequency. The frequency of chromosomal marker transfer in high frequency recombi- nation crosses (Hfr x F‘) is at least 1, 000 fold greater than that ob- served in an F+ x F' cross (Jacob and Wollman, 1961). Both types of male cultures transfer chromosomal markers to an F+ culture at a frequency about one-tenth as great as that observed when F " cells are used as recipients (Jacob and Wollman, 1961). The ability of two male cultures to interconjugate is dependent upon the occurrence of spontaneous Hfr and F- mutants in both cultures. Similarly, the frequency with which an F-factor is transferred to recipient cells during conjugation depends upon the state of the promotor. Cytoplasmic F-factors are transferred with an efficiency approaching 100%. In E. 2E 70% of the recipient cells receive the sex factor within five minutes of the onset of conjugation (Jacob and Wollman, 1961). 10 An integrated F-factor is transferred at a frequency of approxi- mately one thousandth of the frequency of the proximal marker (Jacob and Wollman, 1961). The low frequency of transmission of an inte- grated F-particle is a reflection of (1) its terminal position on the linear chromosome and (2) the probability of random chromosomal rupture during conduction to recipient cells. The frequency with which a specific marker is conducted to a recipient cell depends upon (1) its distance from the origin on the chromosome, (2) the rate of chromosomal transfer, and (3) the resist- ance of specific pairs to spontaneous and operational disruption. When a population of Hfr cells is allowed to mate with F' cells undisturbed for a suitable length of time and is then plated on media selective for recombinants having received different markers from the Hfr, a gradient of transmission of genetic material is observed (Hayes, 1964). This gradient results from spontaneous rupture of thechromo- some during transfer. Assuming that the probability of rupture is constant per unit length of the chromosome and that the rate of transfer of the chromosome from donor to recipient is constant, the observed recombination rate for a given Hfr marker is the negative exponential function of that marker's distance (measured in time units) from the point of origin (Jacob and Wollman, 1961). The direct relation between the distance from the origin and the recombination frequency of an Hfr marker has been experimentally confirmed by Taylor and Adelberg (1960). The genetic polarity of the Hfr chromosome allows a distinction between two categories of genetic characters: those for which the frequency of recombination is high and those for which it is low (Hayes, 1953). That portion of the Hfr chromosome carrying genes of the first category is called the proximal segment. That portion of the Hfr chromosome carrying genes of the second category is called the distal Segment. The existence of genetic polarity in Hfr strains indicates that 11 the Hfr chromosome is a linear, discontinuous structure which is defined by two extreminities: the origin and the Hfr gene (Jacob and Wollman, 1961). The order in which donor markers are transferred is character- istic and constant for the Hfr strain employed. By varying the means of selection, Hfr strains may be isolated which differ with respect to the characters they transfer with high frequency. Whereas one Hfr strain may transfer two markers as if they were located distal to one another on the Hfr chromosome, a second Hfr isolate may transfer the same two markers with a frequency characteristic of proximal markers. These anomolies of transfer are reconciled by picturing the F' and F+ chromosome as a closed, continuous structure which is transformed in- to a linear, discontinuous, transferrable form during mating by random interaction with an F-factor (Jacob and Wollman, 1958; 1961). Genetic Anilysis of a Linkage Group by Conjugation Three methods of genetic analysis have been used to dissect the bacterial chromosome by means of conjugation. These methods and their applications are described briefly below. A more detailed evalu- ation is presented in Material and Methods. Mapping by the Gradient of Transmission-~Genetic characters of an Hfr strain are transmitted to recombinants with different and characteristic frequencies. The genetic constitution of zygotes depends solely on random chromosomal breakage during transfer which is, in turn, dependent upon the distance of the markers from the origin of the Hfr chromosome. The genetic characters of the Hfr strain thus may be ordered according to the decreasing frequency of their transmission (Jacob and Wollman, 1961). Since chromosomal breakage is random, 12 however, this method is also valid for estimating the distances between markers as well as their relative order (Hayes, 1964). MappingEy Genetic Analysis of Recombinants--Using a few appro- priate selections, linkage relationships may be established between unselected Hfr markers and those characters chosen for the selection of recombinants. This method is suitable for mapping genetic characters distal to the selected markers (Jacob and Wollman, 1961). Mapping in Time Units—-Genes on the proximal segment of the donor chromosome may be ordered in relation to the leading extremity by determining the specific time at which each marker begins to appear in recombinants following separation of mating pairs at specified time intervals by means of violent agitation (Hayes, 1964; Jacob and Wollman, 1961). Pictorial Representation of the E. coli and E. tygiimurium Chromo somes The chromosomes of both E. coli and E. typhimurium have been extensively mapped by conjugational analyses. The circular linkage groups of E. coli (Hayes, 1964; Jacob and Wollman, 1961) and E. typhimurium (Sanderson and Demerec, 1964) are presented in Fig. l and 2. Figure 3 indicates regions of homology on the two chromosomes. Occurrence of Intra- and Intergeneric Conjugation Intrageneric conjugation has been observed in the following genera of bacteria: Escherichia (Lederberg and Tatum, 1946), Salmonella (Zinder, 1960b), Pseudomonas (Holloway and Fargie, 1960), and Serratia (Belser and Bunting, 1956). Intergeneric conjugation has been reported to occur between the following pairs of genera: Escherichia-Serratia; Salrnonella-Serratia; thi mal str arg arg (arg A) 13 thr ara Ieu . zi I oro lac 10 min gal (arg) is Figure 1. Positions of some markers on the circular E. coli linkage group. Abbreviations: thr=threonine; ara=arabinose; leu=leucine; azi=azide; pro=proline; lac=lactose; gal=galactose; his=histidine; arg= arginine; R=regulator; str=streptomycin; mal=maltose; xyl=xylose; mtl=mannitol; mot=motility; cys=cysteine; ile=isoleucine; thi: thiamine. Markers whose exact position is uncertain appear in parentheses. l4 str arg G arg E his Figure 2. Positions of several markers on the circular E. typhimurium linkage group. Abbreviations: thr=threonine; ara=arabinose; leu=leucine; azi=azide; arg=arginine; pro=proline; asp=aspartic acid; asc=ascorbic acid; gal=galactose; cys=cysteine; try=tryptophane; his=histidine; str=streptomycin; xyl=xylose; mtl=mannitol; ile=isoleucine; thi=thiam ine . 15 thr str arg his Figure 3. Regions of homology between the E. coli and E. yphimurim chromosomes. Abbreviations: thr=threonine; ara=arabinose; leu=leucine; azi=azide; pro=proline; gal=galactose; his=histidine; arg=arginine; str=streptomycin; xyl=xylose; mtl=mannitol; thi=thiamine . l6 Shigella-Salmonella; Escherichia-Salmonella (cf. Clark and Adelberg, 1962); and Escherichia-Proteus (Falkow e_t_ a_l. , 1964). Conjugation between Escherichia and Salmonella has been shown to occur in a mammalian host (Falkow and Baron, 1962). In most of these cases, however, evidence that the observed genetic transfer requires or even involves cell contact is incomplete (Clark and Adelberg, 1962; Miyake, 1962L MATERIALS AND METHODS Cultures The bacterial strains employed and the genetic characteristics of each which are pertinent to this study are listed in Table 1. Various mutant markers and sex factors were added to Salmonella pullorum 35W as required. These mutants as they appear in the text are designated 35 followed by the mutant genotype. Forty-seven additional strains of E. pullorum were employed to survey for donor ability. The strains surveyed included E. pullorum 1-38, 43, and 45-54. Nineteen bacteriophages isolated from lysogenic strains of E. pullorum (Robinson, unpublished) were used to study promotor in- duced alterations of the male cell surface. These bacterioPhages are designated: 1, 4, 5, 6, 12, 15, 16, 22, 24, 26, 29, 33, 34, 36, 37, 38, 40, 46, and 50. Media A list of the media used and the purpose of each is given in Table II. Composition of Synthetic Media Minimal E Medium ('Vogel and Bonner, 1956) MgSO4. 7HZO 0. 2 g Citric acid. H20 2. 0 g KZHPO4 anhydrous 10. 0 g NaHNH4PO4. 41-120 3. 5 g ' Carbohydrate 4. 0 g Distilled water 1000 ml 17 18 .udmumfimohnu mo>fizmaomnm 333305.305 “coflodooum goofing: "couscoum no oouwdus you" n "voodoofim no .oouflfidu... “HofifidenflE momoaxnaxmomocfianmHmum "occuoddmmufimw momouoddnomfi ”2:33??de 3583533» “camcoounp ".23 mocwcofifiognuog nocdododnsg modfioumnoua nonwoumcnonmlwo "3038.13 one mcofimgounnafl. m + + + + + + .3“. undrm mw>m< m + + u . uzuusflnufi 1m 2: u .. u n ma a a do u a + + + .. .£+.£+ H+ a 2m m2fl< H + + - - - - -oa-Ee-:£-ufi 2m 33... m + + + + + + Lfindofiuufi 1h «A: m + + + + + + Loan +h c3 300 «Eofiuonomm m .. .. + + + + ionmifioaumewo A+pmv @ u u u > Soho dd .m 0:05 m m 3.”: Tax mu+m 1mm 03 tomato 0 km 3mm 2 2 H m .Sm «32 ES cofimuflfid muouomumgo 09$ Eek—m on. uncommom oudnp>£onnm0 0230:9314 wcflmz . magnum anion—own. mo mofimwnouoduQEUuu .H HAm£onnmo sand comm caduofwaonnmo 05v .0 ”55305 Hmflcouomfio oofiQ H393. con #98an GOSMNSSS coon—odd Aomfiumgmv Hmw< mowom ocfigmmmo cam .0 ”55308 Hmficouowfio oofiQ mzm winged Amoudfidu made we Gofiuoouop moudfido 3mg >Houmuondd A02”: MOM ooufigov cumuolzsonumo §w .0 503.333: vacuolwaonndo Hmowmofiofim ongomfi ”305.35 mgom OGMEdmmo Row .0 "55:58 Hmfioouomfio ouochfidm mzm what/04 5:355 AuBOHm 0030 flown 533.56% mucogofimmsm omomudm condom 53:52 .533 m0 omomusm of one cow: ozone mo um: H 3an com mug 300m moan 55365 H fioun ”33.3.3“ >\> mmmm. .0 mafia 55305 H mcoUooHom 0330mm new 5.9335 H 3 wooed mucogoamdm new >H 0.238 new mudmcwngooou mo dogwoouoo "5:255 ozuooaom Enzyme fife/cum mama: omega mo comuoou now new E5305 AHBOHm mudwudg oofiooaofi: can Eon 055m mo cowumofimficog mundane: oofioofios: one Bod 058d ugfiwpofi Hmflcouomflo mudmfingooou mo comuoouov "gfimfivg m>wuuvfivm E5258 :3 unnamed? £93 0-2 Home oGOfiEAHH a; s38 Hood m UOQUMHGU ”Anson asses m 3.552 21 Minimal E. Agar--Double strength agar (15 g Bacto-Agar per 500 ml distilled water) and double strength E salts were autoclaved separately and mixed when both solutions had cooled to 45C. Pool Media--Nine pool media were prepared by supple- menting minimal E agar with 10 ml/liter of amino acid and purine and pyrimidine pools 1-9. The composition of pools 1-9 is given in Table III._ Watanabe (W) Medium (Watanabe and Watanabe, 1959). KZHPO4 0. 3 g KHZPO4 0.1 g NH4C1 0. 5 g NazSO4 0.1 g MgSO., 0. 2 g Carbohydrate 0. 5 g Distilled water 1000 ml Soft Agar Bacto-Agar 7. 0 g Distilled water 1000 ml Composition of Complex Media T ryptone Agar Bacto-Tryptone 10 g Bacto-Agar 10 g NaCl 8 g Dextrose 5 g N CaClz 0.1 ml Distilled water 1000 m1 .H8\w8 m .o n m5805> £8\m8 N n m05388>d can 00553 can 5500 0580 ”080801530 m0 080303800800 5 s: :m m 5.5.... is. 22 * 500 500 055me 58007010 05:50 0503m 589.0% 0 05180 053mm 088005: 0805808980. 0500.53 118053 w 059: 00:01? 05050505 5.008: 050358 h 0585:» 0585508 05853 058.5% 0500500 0 m w m N H .08 Hoonm a 500m 05585.?»Q can 055& one 500 0580 Ho 8055009800.. A: Mdm<fi a. 23 m o I I + : H + I + + I I .30 0: I + + .. + I I I + 3 u + I + I I .30 02 I + + + + I I + o H + . + + I I + I .30 m>0+mfi£ I + I + I I I w H + I - + + + + + * HHS I I + l I N. + I - + + + + + 135 I I I I 0 + + + - + o m - - - + + + 0 1.. - - - - m + + + - I + + + + + m «E I I .. w. Hue +H + + + I + > + I .. + + m n m H x I I I H u + + + + I + m + - - + + N m .m I I 0.3 +H + + I .. - + - - + - Hum 1.3 .2: I + I I + a H + + + + I + I a I I + HOD—g uuum+mws ounm 0: .904 RAH. 9A0 mam “AH. 32 H X 80,302 .Sm 0.20%ng o o I o w H m m H m “ 2.4 .00300m 00.000muH00HH Ho HH0> 0 00000 0033 0200 m0HNHHH0HH000 000 00000 HHHOHH H00: 0005 w0HH0H00000HHH0 00H00H00 0m00H 000000HH 00000>HH|0HH000 0H3 w0HNHIHH0HH 00 030000 0HH00 .000H0000 0H5 >3 0000300000000 000 0H 00000 000 0000000 HH00 .H 0H00: §00HHHHm .m 0H HH .Omw 0» 00H000 00: 00.30000 0H0 0003 w0H>0H00000 0030 00000 0003 000000>HHOHH000 000 000300 0H0>0000m000m .m0H>0H00000 00 0300 3000 E 030000 03000 00 00000 0003 0300 00.3000 000 00000003900 0H000HH> 000 0H00 00HH00H000nH0000 ”$0.0 000000>HHOHH000 “Hafiwwim 00HH0000H> “Hfidom H0 003000000000 H00HH 0 0>Hm 00 00000 0003 0000000396 0H00 00HG00 "00000001500 00 000Hu000000000+ 00000000 00 00NHHH00 000 n I “0000000HH 00 00NHHHHHH n+ "00000H00 000x005 00H "00000 000 u I H00000u+ m0>HHH0000u0 "000000H000n0 H00HN0HHN0 H0H0>0000HH0000H000 ”00HH00HHn00HH n00H000H00Hn0HH “00H000Hn00H H00H0000HHun0HHH H00H000>0n0>0 H00H0H00HHH "0IHHH n00H000H0HHnHHHH “H00H000H0HH000 M000H>an>x M000000H0wnH0m "00000000530 ”300,050 000 H.000H00H>00HH0.¢IVon H00 00& I I I I I I. I .H. + + + + + + + 3 H00 0 H I I I I I I I .H. + H. + + + + + , + 3 H00 0HHH + I + + + I + I I I I I H + + 2 I30 00 0H: I + + I I .H + + + + I I I I .. + 0H Reagents 25 M-9 Broth (Vaughan, 1962) 2X M-9 Salts—-Components were dissolved separately and mixed. KHZPO4 , 6 g NazHPO4 12 g NH4C1 2 g Distilled water 900 m1 2X Casamino Acids Difco Casamino Acids 30 g Distilled water 1000 ml Norite A activated 2 teaspoons charcoal The solution was allowed to stand overnight at 4C, and was then filtered, and autoclaved. Preparation of M—9 Broth 2X M-9 salts 500 m1 291: Casamino acids 500 m1 1M MgSO4. 71-120 2. 5 ml 25% NaCl 2.0 m1 40%Dextrose 5. 0 ml The production of indole was detected in SIIVI agar stabs by over- laying the cultures with 0. 5 ml chloroform followed immediately by 0. 5 m1 Kovac's reagent. A deep red color formed in the chloroform layer when indole was present. Kovac's Reagent Am yl alcohol 7 5 ml Hydrochloric acid 25 m1 (concentrated) p-Dimethylaminobenzaldehyde 5 g 26 Mutation Proc edures Induction of Mutations with Z-Aminopurine--Approximately 100 cells of a logarithmic culture of the strain to be mutated were inocu- 1ated into 10 m1 nutrient broth containing ZO‘ug/ml cysteine and ZOOug/ml Z-aminopurine. The culture was incubated at 37C with aeration until maximum turbidity developed (Hartman it a_._l. , 1962). Isolation and selection of mutants Isolation of fermentation mutants--Mutants no longer cap- ‘ able of utilizing specific carbohydrates as carbon sources were de- tected by plating 103-104 cells of a Z-aminopurine treated culture on eosin-methylene blue agar plus 0.4% galactose (EMB-gal agar), EMB- mtl agar, and on phenol red agar plus 0. 4% xylose (PR-xly agar). Plates were incubated at 37C for 48 hours. When EMB agar was used as a differential medium, the mutant colonies appeared white, dark red, or white with a small dark red center. The mutant phenotype was readily distinguishable from the brilliant green sheen character- istic of fermenting colonies. When phenol red agar was the differential medium, nonfermenting mutants produced red colonies in contrast to the yellow fermenting colonies. Selection of amino acid and nucleotide mutants by the penicillin method--Mutants deficient in the biosynthesis of amino acids and nucleotides were selected by the penicillin screening method (Davis, 1948; Lederberg and Zinder, 1948; Gorini and Kaufman, 1960). According to this method 0. 01 m1 of a washed Z-aminopurine treated culture was inoculated into each of two tubes containing 3 m1 E medium supplemented wichO‘ug/ml of cysteine, leucine, and any other amino acids required by the wild type strain. A third tube was inoculated with O. 1 m1 of the mutagen-treated strain. 27 After 60 hours incubation at 37C without aeration 0. 1 and 0. 5 ml of the contents of each tube were spread onto double enriched E medium. Small colonies, some barely visible, were transferred with sterile toothpicks to nutrient agar plates in a template fashion with 24 colonies per plate. After 48 hours incubation at 37C the nutrient agar master plates were replicated by the velveteen method (Lederberg and Lederberg, 1952) to nine pool agar plates. The plates were incubated at 37C for 48 hours. The mutant lesion expressed by each colony was indicated by determining the amino acid or nucleotide common to the pools on which the mutant grew. The requirement of each mutant was confirmed by a crystal test as follows. The growth from a pool agar plate was trans- ferred to 3 ml soft agar and poured on to the surface of E agar supple- mented with the requirements of the wild type strain. A few crystals of the suspected requirement were placed on the surface of the agar overlay. If the added supplement were indeed required, growth was observed only within the area of the crystals. In the event that no mutants were found following 2-aminopurine induction, the treated culture was diluted 1:100 in fresh nutrient broth + cysteine + Z-aminopurine, and the entire procedure was repeated. Often during the course of the study a specific type of mutant - e. g. a tryptophaneless mutant - was required. In such cases an enrichment procedure preceded penicillin selection. A Z-aminopurine- treated culture was washed with saline and diluted 1:10 in E medium to which had been added all the supplements required by the wild type strain plus the amino acid required - e. g. tryptophane - by the type of mutant sought. The enrichment culture was incubated at 37C with aeration until maximum turbidity was reached. This process allowed growth of only wild type and the particular kind of mutant being selected, thereby enriching the proportion of this mutant in the culture. 28 The enriched culture was washed with saline, and the penicillin selection method described above was followed. Selection of amino acid and nucleotide mutants by thymine- less death--Polyauxotrophic mutants of a thymineless strain resist thymineless death in the absence of the required amino acid because they are not able to initiate new rounds of abortive DNA replication (Maalgfe and Hanawalt, 1961). Thymine starvation may thus be used to select doubly auxotrophic mutants (thymine' amino acid') in a mutagen- treated thymineless population (Wachsman and Hogg, 1964). To initiate thymineless death 1. 0 ml of a washed, 2-aminopurine- induced suspension of _S_. pullorum 35 his-ara-xyl-thymine- was trans- ferred to 9, m1 E medium supplemented with cysteine, leucine, histidine, 0.4% glucose, and 1. 3% sucrose. The culture was incubated at 37C without aeration. Samples were withdrawn at 12 hour intervals, diluted 103, and plated on double enriched E medium supplemented with 200g thymine, histidine, cysteine, and leucine/ml. After 48 hours incubation at 37C the plates were scored for large (prototrophic) and small (possible auxotrophic) colonies. - Detection of Male Cultures Altered Surface Properties--Two criteria were used for the detection of the cell surface alterations which characterize donor bacteria: (1) permeability to eosin dye (the staining reaction), and (2) insusceptibility to a female specific _S_. pullorum bacteriophage. The Staining reaction- -A test of staining reaction consisted of spotting logarithmic cultures (penassay broth) of the bacteria to be tested on eosin-methylene blue agar without fermentable sugar (EMO agar) (M51015 e_t a_._l. , 1962). The plates were incubated 6-12 hours at 37C followed by incubation at 25C for a period of 1—7 days. Male cultures 29 take up eosin dye from the medium and the spot of growth appears pink to red when examined by reflected light. Female cultures remain white. Each culture tested for a staining reaction was spotted beside a known F- culture of the same genus, species, and preferrably, also the same strain. The time of incubation at 37C and at 25C is a critical factor in evaluating the staining reaction and must be standardized for each culture. Several generalizations about the time-temperature relation- ship can, however, be made. Incubation for 10-15 hours at 37C followed by 24-48 hours at 25C is usually sufficient to distinguish positively from negatively staining E. _c_c_>_1_i cultures. Rapid growing strainsof E__. 52.1.1 - e.g. 12. c_9_1_i 107- on the other hand, require as little as 6 'hours incubation at 37C only. S_. pullorum cultures require 24 hours incubation at 37C followed by 2-4 days at 25C. Slow growing _S_. pullorum strains - e.g. _S_. pullorum 35 chloramphenicol resistant (Cmr) and 35 F'lac+Cmr - require 24 hours incubation at 37C followed by one week at 25C. In the case of rapidly growing strains, the spots were observed for a color differential every 2 hours. In general, however, plates were examined after 15 and 25 hours at 37C and each day at 25C for seven days. If no color difference between the control and test spots was observed at the end of one week at 25C, the test cultures were assumed to be F-. On prolonged incubation at either 37 or 25C both male and female cultures gave a positive staining reaction. Thus, it was always a clear color differential which was sought. A staining reaction may also be detected in isolated colonies. A positive color, however, requires longer times of incubation at 37 and 25C to deve10p. A positive staining reaction in a colony was 30 always confirmed by a spot test~ of a logarithmic culture grown from that colony. Insusceptibility to a female specific S. pullorum bacterio- MuNineteen bacteriophages previously isolated from lysogenic strains of S. pullorum were tested for an ability to distinguish between male and female strains of S. pullorum. Each phage was spotted by loopfuls at its critical test dilution (Adams, 1959) onto S. pullorum 35 F- and onto 35 F'lac+gal+, 35F+Wf> and 35 Hfr-1 in tryptone soft agar I overlay on tryptone agar plates. After incubation at 37C for 1‘0-12 hours the spots were scored for lysis. Phage 15 was found to be female specific (see results). Phage 15 was spotted on cultures of S. pullorum suspected of being donors in tryptone soft agar. Incubation and scoring were performed as described above. + .. Transfer of sex factors (Tube matiggmethod)--F and F strains were tested for an ability to rapidly donate sex factors to female cul- tures by the tube mating method. According to this method donor and recipient strains were added to 10 ml penassay broth in a ratio of 1 male to 10 female cells (Jacob and Wollman, 1961) for E_. £215;- E. coli and for E. (213 - S. pullorum crosses to give a final concentration of approximately 2x107 bacteria per ml. The mixture was incubated with- out aeration for 10-15 hours. A ratio of one donor to one recipient cell was used forS. pullorum - _S_. pullorum crosses. The mating mixture was then diluted to give 100-1000 colonies per plate and was plated on a medium to contraselect the donor and/or differentiate male and female colonies. + - Transfer of chromosomal markers in F x F crosses—-A quali- tative tube survey method was employed for examining a large number of cultures for an ability to conduct chromosomal markers. A series of 13x100 mm test tubes containing 1 ml penassay broth was inoculated 31 with various cultures to be tested. The cultures were allowed to attain the logarithmic phase of growth (3-4 hours at 37C). One ml of a logarithmic culture of recipient in penassay broth was added to each tube. The tubes were incubated at 37C for 4 hours to permit genetic transfer and zygote stabilization. After the incubation period 100pfuls of the mating mixtures were spotted on media contraselective for the donor and selective for specific recombinants. Plates were incubated for from 48 hours to 4 days, depending upon the selective medium employed. This same method was also used to survey F+ and F- strains for an ability to donate and receive fertility factors. When chromosomal transfer was to be more precisely determined Imatings were carried out by the flask- shaker method. For matings in which _S_ pullorum was the donor strain and _E. c_ol_i the recipient one ml of a logarithmic culture of S. pullorum was transferred to 50 ml penassay broth in a 500 ml Erlenmeyer flask and incubated on a recipro- cal shaker at the slowest speed that can be maintained until the log- arithmic phase was reached. Five-tenths ml of an exponentially growing culture of §.<£1_i_ recipient was added to the flask. The mating mixture was incubated at 37C for 12 hours with gentle shaking. The culture was then washed twice with saline and concentrated five fold. One-tenth m1 samples were plated on media contraselective for the donor as above. When strains of _S_. pullorum were used both as donor and as recipient the inoculation procedure was reversed. One ml of recipient was transferred to 50 ml penas say broth and was grown as above. When the recipient reached the logarithmic phase of growth 5 ml of donor culture was added to the flask. Reversal of the incubation pro- cedure was necessary to obtain the proper ratio of donor to recipient cells. + Curing of F and F' cultures with ac ridine dyes--Stock solutions of ac ridine orange and neutral red containing 1 mg/ml distilled water 32 were prepared immediately before each curing experiment. The tubed dye solutions were wrapped in aluminum foil and autoclaved at 15 pounds pressure for 20 minutes. The acridine dyes were kept in the dark at all times, and all operations prior to the final plating of treated cultures were performed in the absence of white light. Increasing quantities of the stock solutions were added to a series of tubes contain- ing 5 m1 nutrient broth adjusted to pH 7.6 and supplemented with 200g cysteine/ml according to Table V. One-hundredth ml of a logarithmic culture (penassay broth) of the strain to be cured was transferred to each tube. After incubation in the dark at 37C for 24 hours cells from each tube were streaked for isolated colonies on EMO, EMB-lac, or EMB-gal agar, depending upon the F-factor being removed. EMO plates were incubated at 37C for 24 hours followed by 1-7 days incubation at 25C. EMB-lac and EMB-gal plates were incubated at 37C for 48 hours. Preparation of Mating Mixtures for Electron Microscopic Examination Negative Staining with Phosphotungstic Acid (PTA)--A donor and recipient strain were grown separately in penas say broth, centrifuged, and resuspended in M-9 broth. The two cultures were mixed in a ratio of 1:1 v/v and incubated at 37C for 30 minutes. At the end of the incu- bation period the undiluted mating mixture was mixed 1:1 v/v with 2% PTA. The stained cells were immediately dropped onto formvar covered copper grids with a pasteur pipette. The grids were left un- disturbed for 5 minutes. Excess stained culture was removed by touch- ing a piece of filter paper to the edge of the grid. Grids were allowed to dry at 25C. Positive Staining with Uranyl Acetate (UA)--A 1:1 mixture of a donor and recipient culture prepared as above was dropped onto formvar 33 + TABLE V. --Concentrations of acridine dyes used for curing F and F' cultures . Concentration of dye Ml stock solution/ in ug/ml 5 ml medimn 0 0.00 2 0.01 4 0.02 8 . 0.04 10 0.05 12 0.06 16 0.08 20 0.10 32 0.16 40 0.20 60 0.30 80 0.40 100 0.50 34 covered grids. After standing undisturbed for 5 minutes the grids were floated specimen side downon the surface of distilled water in a watch glass for 5 minutes to remove precipitable nutrients. The grids were then transferred to the surface of 0. 5% uranyl acetate in a watch glass for 60 seconds, removed to dry filter paper, and allowed to dry at 25C. Shadow Casting--Grids prepared as for UA staining were fixed by exposure to the vapors of 2% osmic acid for 2-3 minutes and were then shadow cast with tungsten. Stained and shadow cast specimens were examined with an electron mic rosc0pe. Isolation of High Frequency Recombination (Hfr) Strains A low frequency recombination culture was grown to the log- arithmic phase in penassay broth without aeration, centrifuged, resuspended in an equal volume of saline, and diluted to a concen- tration of 2x108 cells/ml (0. 086 O. D. at 420 mp in a Spectronic 20). The diluted culture was irradiated with an ultraviolet lamp at 20 inches for 20 seconds to give 1% survival. One-tenth ml aliquots-of the irradiated culture were spread onto nutrient agar plates. After incubation in the dark at 37C for 48 hours the nutrient agar plates were replicated to selective agar which had been spread with 0. 1 m1 of a washed exponential culture of recipient concentrated ten-fold. The replicate plates were incubated at 37C for 4 days. Areas on the donor plates which gave rise to recombinants on selective agar spread with recipient were transferred to penassay broth and incubated at 37C for 12 hours. These possible Hfr cultures [were diluted to give approximately 2000 and 200 colonies when plated on nutrient agar plates. The replication process was repeated. 35 An isolated colony which gave rise to a recombinant colony on selective medium plus recipient was grown to the logarithmic phase in penassay broth. The dilution and replication processes were repeated two more times. MatiggProcedures for Hfr x F- Crosses Donor and recipient strains were grown to the exponential phase in penassay broth without aeration. The donor culture was diluted to a concentration of 1x108 cells/ml (0. 05 O.D. at 420 mp). The recipient culture was diluted to 2x108 cells/ml (0. 086 O. D. at 420 mu). The actual number of viable cells per ml of the donor and of the recipient was determined by plating a 106 dilution of each culture on nutrient agar plates. Five-tenths m1 of the diluted donor culture was added to 4. 5 ml of the recipient culture in a 125 ml Erlenmeyer flask. The mating mixture was incubated in a‘water bath at 37C without disturbance (Hartman e_t a_l. , 1962). Gradient of Transmission--The gradient of transmission of genes on the_S_. pullorum Hfr chromosome was established by plating 10'1 to 105 1‘ dilutions of a mating mixture after 90 minutes uninterrupted incu- bation at 37C in soft agar overlays on various selective media (Jacob and Wollman, 1961). Plates were scored for the number of recombinants per 100 initial Hfr cells after 72 hours to 7 days incubation at 37C. Magaping in Time Units (Interrupted Mating) Siringe method of interruption—One or 1. 5 ml samples were withdrawn from a mating mixture at 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, 120 minutes unless otherwise stated and were transferred to cold sterile serum bottles. Each sample was drawn up and expressed ten times with a cold 2 ml syringe fitted with a 26 guage needle in order to rupture the conjugal bridge and, therefore, the chromosome 36 (Kitsuji, 1964). The interrupted mating samples were transferred to chilled sterile centrifuge tubes, sedimented at 9750 g for 15 minutes, and resuspended in an equal volume of cold saline. Samples were kept cold at all times to prevent chromosomal transfer (Jacob and Wollman, 1961) and specific pair formation (Clark and Adelberg, 1962). One-tenth m1 aliquots of 10'? 10’,l 10',2 10',3 and 10-4di1utions were added to 3 ml soft agar and were overlaid onto various selective media. Plates were scored for the number of recombinants per 100 initial Hfr cells after 72 hours to 7 days incubation at 37C. Waring Blendor Method of IntermtionnOne-tenth ml samples were withdrawn from a mating mixture at specified time inter- vals and were transferred to 9.9 ml of cold saline. The diluted mating mixture was poured into a 30 ml capacity Waring blendor cup and blended for 2 minutes at 4C. One-tenth ml aliquots of 10'? 10", 10'2 dilutions of the mating mixture were transferred to tubes of soft agar and were overlaid onto selective media as described in the preceding para- graph. Plates were scored for the number of recombinants per 100 Hfr cells after the required incubation period. Genetic Analysis of Recombinants--Individual recombinant colonies from a given selection were transferred to form the pattern of a grid, regularly arranged on the surface of a plate containing the same medium as that used for their selection. After 48 hours incubation at 37C these plates were replicated to plates containing a variety of selective media which allow the determination of the genetic characteristics of the re- combinants. In any selection 100 recombinants were analyzed per time sample. After incubation at 37C for 48 hours the plates were scored for the percentage of the selected recombinants which had received each of the genetic characters analyzed from the Hfr donor (Jacob and Wollman, 1961). RESULTS Induction of Mutation with 2-Aminopurine and Selection of Mutants 2-Aminopurine was found to be an excellent mutagen for S. pullorum 35. There was no specificity with regard to the markers mutated. A brief list of the mutants isolated following Z-aminopurine induction is given in Table VI. Figure 4 presents the data on selection of amino acid and nucleo- tide mutants by thymineless death. Thirty-six hours incubation of a mutagen treated culture in synthetic medium without thymine permits detection of the largest percentage of doubly auxotrophic mutants. Survey of S. pullorum Bacteriophages for Male or Female Specificity Nineteen bacteriophages isolated from lysogenic strains of _S_. pullorum were surveyed for an ability to distinguish male and female strains of S. pullorum. The results of this survey are presented in Table VII. Phage 15 at its critical test dilution (2. 7x105 phage/ml) produced no lysis or very few turbid plaques on male strains but caused con- fluent or almost confluent lysis of female strains. This phage was used to confirm the maleness of F+, F' , and Hfr cultures of _S_. pullorum. + _ Phage 15 caused no lysis of F or F- strains of _E coli W6. Survey of S. pullorum Strains for Maleness The most readily detectable criterion of maleness in bacterial strains is a positive staining reaction. Forty-nine strains of_S. pullorum 37 38 TABLE VI. --Types of mutants isolated following 2-aminopurine induction Type of mutant of _S_. pullorum 35 No. of mutants ara- 24 ara sens... 1 try- 1 thr‘ 1 35PM“D gal- 1 3151930”6 mtl- 1 Hfr - 1 mtl- 1 Hfr - 1 ilva- 1 his” 1 his- ara- 4 his- ara- xyl- 20 h'is- ara- xyl- pro- 2 his: ara' xyl: thy'_ 1 his_ ara: xyl_ mtl_ 4 his_ ara_' xyl_ ade_ _ 1 his ara xyl mtl thr 1 his- ara- xyl- mtl- ade_ 1 his- ara- xyl- mtl- gal- 2 his- ara- xyl- mtl- gal- ile- 1 his- ara- xyl- mtl- gal- ile- pro- 1 his- ara- xyl- gal- mtl- dex- 1 Abbreviations: ara=arabinose; sens.=sensitive; try=trypt0phane; thr: threonine; gal=galactose; mtl=mannitol; ilva=isoleucine + valine; his=histidine; xyl=xylose; pro=proline; thy‘xthymine; ade=adenine; ile=isoleucine; dex=dextrose. Log no. Survivors or Possible Mutants/ml. 39 Time (hours) 7 a 3. 7.:- 64... . q. 12 5" ".10 4.. «r8 ..6 30- .o o . log no. survivors K K " log no. possible mutants 2-- ‘~ ‘5 ‘ percent possible mutants "4 Q ‘ 1T “2 Figure 4. Selection of mutants by thymineless death '. in S_. pullorum 35. 0- .' E .L i. 4. 4 I : : 0 10 20 30 40 50 60 70 80 90 100 Pe rc ent Possible Mutants/m1 .000Ho0HHH 00m 300 >00> u Hoax? H000UOHHH H Hum 40 “0H0>H 000G000 000000 N H00 H0H0>H 00003000 .I. H0 H000H00H>00nn< I H0 H0 H.0 H0 H00 H0 H0 om I H0 H0 H0 H0 H0 H0 H0 00 I H0 H0 H0 H0 H0 H0 H0 o0 I H00 Hon HXH H0 H00 H00 H0 mm I H0 H0 H0 H0 H0 H0 H0 pm I H0 H0 H0 H0 H0 H0 H0 cm I H0 H0 H0 H0 H0 H0 H0 Hum I Ho Ho 00 Ho Ho Ho Ho mm I Ho Ho 00 Ho Ho Ho Ho 00 I H0 H0 H0 H0 H0 H0 H0 on I H0 H0 H0 H0 H0 H0 H0 0N I H0 H0 HXH H0 H0 H0 H0 NN Hoax? I #0 van/00.00% E???Vim/£00000/ H I Hoax? Hem 0915 H0 Um H00 H0 0 I H0 H0 HXHVHB H0 H00 H0 H0 m I I I I I I I I 0 I H0 H0 H0 H0 H0 H0 H0 H 0m0HHnH 00000 00000 00000 H0000HH0HH .m 0m 00 . 0 0m 00 o 000 0 +H + H.....H H a 03+hmm 0 +H + Him 03+hmm mm H . Hm >HH0HHH00Q0 X00 000 00m0HHnHoH00000n H0000oHH0m .Mmo 00:30-- .HH> ”.3000. 41 were examined for permeability to eosin by spotting them on EMO agar. Forty-eight of these cultures gave a negative staining reaction thus placing them in the category of F- or F0. One positively staining strain, S. pullorum 6, was detected. This strain was subsequently shown to contain a sex factor (see below). However, because of its specificity with regard to the nature of recipients with which it will mate _S_. pullorum 6 was not initially thought to be appropriate to the establishment of a conjugation system in S. pullorum. Transfer of Sex Factors to S. pullorum In the apparent absence of a suitable naturally occurring male it was necessary to transfer donor ability to S. pullorum from male strains of _E_. £93. Two types of sex factors were introduced into S. pullorum 35: (1) the classic F+ element to permit isolation of Hfr strains with various origins, and (2) an F'lac+ episome for the isolation of an Hfr strain with a predictable order of entry of genetic determinants. + Introduction of an F+-Sex Factor into S. pullorum 35W--An F - sex factor was introduced into _S_. pullorum 35W from E.C£1_i_ W6 by conjugating the two strains according to the tube mating method. At the end of the mating period the conjugation mixture was diluted and plated on Levine's EMB agar to give approximately 1000 colonies per plate. Three types of colonies resulted: (1) W6 cells produced large lactose fermenting colonies. (2) S. pullorum 35 cells produced small white nonfermenting colonies and (3) small pink (staining reaction+) nonfermenting colonies. The small pink colonies were transferred to penassay broth, grown to the logarithmic phase, and spotted on EMO agar to confirm their permeability to eosin. Twenty-eight percent of the small pink S. pullorum colonies stained positively in the confirm- . . . . . . + atory test of staining reaction. These staining reaction cultures were 42 tested for an ability to ferment lactose, an ability to produce indole, and motility. Most of the positively staining cultures possessed the chromosomal genotype of the recipient strain (lac-ind-mot-L The S. pullorum 35 staining reaction+ culture which gave the strongest staining reaction was identified as a possible F+ strain and was designated S. pullorum 35F+W6, indicating its donor ability and the origin of the sex factor it contains. The intensity of the staining reaction in S. pullorum 35F+W6 was initially weak as compared to that observed in F+ §.<£1_i_ cultures. As the strain was transferred, however, the intensity of staining gradually increased, suggesting either a host modification of the sex factor and/or a complex nature of the alterations induced by the newly introduced promotor. + The transfer of an F sex factor to S. pullorum 35W is sum- marized in Figure. 5. + + Introduction of an F'lac Episome into S. pullorum--An F'lac episome (the F-promotor with the lactose region of the _E. 99E chromosome attached) was introduced into a streptomycin resistant mutant of S. pullorum 35 from S. 921i AB785 by the tube mating method. The donor was contraselected by plating approximately 1000 cells from the mating mixture on Levine's EMB agar plus streptomycin. Thirty- three percent of the resulting colonies were phenotypically lac+mot- ind strrstaining reaction+. The stability of the lac+ marker in various S. pullorum 35 strr lac+ isolates varied. Six percent were unstable yielding 1 lac-:5 1ac+ colonies. The remainder segregated 0.05-3. 0% lac- cells. The lac- segregants stained negatively on EMO agar and could be reinfected + with the same F'lac particle (see below). Thus, these variants had . . + . arisen through loss of the entire F'lac particle. 43 Donor Genotype IS. 52E W6 F+lac+mot+ind+staining+ x Recipient F’lac-mot-ind-staining- S. pullo rum 35W I + - - - + S. pullorum 35 F lac mot ind staining , + Figure 5. Transfer of an F sex factor to S. pullorum 35W. 44 The same F'lac+ episome was transferred to S. pullorum 35W according to the tube mating method. Brilliant green agar was used to contraselect the donor and to differentiate 1ac+ and lac- exconjugants. The purpose of this transfer was to provide an F'lac+ donor bearing no antibiotic resistant markers thus permiting its ready contraselection in future matings. Sixty-five percent of the surviving colonies had received an ability to ferment lactose. These fermenting cultures were tested exactly as were the lac-kstrr S. pullorum 35 strains and behaved in all cases as did the majority of the strrlac+ cultures with regard to an ability to transfer a sex factor, to be cured, and to conduct chromosomal markers. For this reason only the data concerning S. pullorum 35 strrlac+ cultures are presented in Results. + Evidence of Donor Ability in S. pullorum 35F W6 Transfer of a sex factor by S. pullorum 35F+W6--A second criterion of maleness in bacterial strains is the ability to transfer fertility factors to F- cultures, producing positively staining colonies of the parental recipient. S. pullorum 35F+W6 was incubated with an E. c_:_o_1_i_ female strain, 104, according to the tube mating method. Approximately 300 cells from the conjugation mixture were spread on each of several EMO agar plates. Thirteen percent of the resulting large pink nonfe rmenting colonies stained positively, indicating that they had received the genetic determinant of permeability to eosin. The ind+mot+staining reaction+ isolate which gave the strongest stain- ing reaction was designated @3213 104F+35. The positive staining reaction was readily lost from E. coli 104 F+35 during early passages of this culture. 45 Similarly the positive staining reaction could be transferred to IE. coli W6 which had been converted to an F- strain by treatment I+W6 with acridine orange (see below) from both S. pullorum 35F and + E. coli 104F+35. These W6F strains transferred the staining re- action to 12.9911 104. S. pullorum 35F+W€J also transferred the ability to stain to a streptomycin resistant mutant of S. pullorum 35W with an efficiency of transfer of approximately 3%. W6F+ and 104+ cultures which received an F-element that had been passed through S. pullorum produced a reduced staining reaction on EMO agar, implying a host modification of the F-element in S. pullo rum . + . . . . Removal of F Sex Factors With Acridine Dyes--As a third criterion of maleness attempts were made to dissociate the positive + staining reaction from the F strain of S. pullorum 35, IS. coli W6 W6 ), and culture 3 + which had received the F-element from S. pullorum 35F W6 by + + (the source of the F element in S. pullorum 35F treatment with ac ridine dyes. The results of treatment of S. pullorum 35F+W6, S. pullorum 6, §.co_1i_ W6, and E. c"__c_>E104F+35 with acridine orange are given in Table VIII. Ac ridine orange was effective in removing the F+ element from E. 3911 strains but was ineffective in removal of an F+ element from S. pullorum because of its toxicity for this organism. Two approaches to the problem of curing S. pullorum were investigated: (1) lowering the concentration of the dye below4Iig/ml and (2) searching for an ac ridine dye which would cure but would be less toxic to the S. pullorum strains. The data in Table IX representstreatment of S. pullorum 6 with low concentrations of acridine orange. The staining reaction was dissociated from S. pullorum 6 within a range of 2. 6-3.0‘Iig ac ridine orange/ml. However, because of the narrow range of curing and the 46 000300 00000 000000HH 0H3 0H00H00H 00090002 .0. £0300w 00 u OZ “m0H000 00 n 02 H000H00H>000h< A DZ 07H OCH OCH vwooH A 02 o 00000HH0m .WI 0H00Hm 0.0 w.m 05 Him N.m o.m w.m ed 00 N.N o.~ OH 0 HH..HH\.mi 0H 0m000o 00H0H0 00 Ho 003000000000 .0w000o 00H0H000 Ho 0003000000000 30H 5H? 0 00000HH0m .m Ho m0H00UII .KH MiHmme. 000200 00000 H00000m 000 00005 00000002 .0. 5300m 00 u 02 uw0H000 00 n OZ ”00030300394 4 OZ OZ IAJ DZ 02 0 DZ N 0 OH oH 0H m w OZ IM OZ 0 m m m m A OZ 00.00% .w. +03 IEsISHHMm ..0. +003 08 .0 03 :8 .0 0005 o? mm 2: cm oo 00 Nm om 0H NH 0H m H0 N o H0.H\w1 0H 0m000o 0000 00 Ho 003000000000 .0m000o Q0H0H000 HHHH3 00H0000 +.m 00 II 0.030-- .0; 040,00. 47 probability of dilution error when using low levels of dye consistent results were not obtained when the procedure was repeated. A second acridine dye, neutral red, was tested for efficacy of curing (see Table X). - Neutral red proved to be less toxic to _S_. pullorum strains than acridine orange and was an effective curing agent for both _E_. c__ol_i and S. pullorum. Neutral red in no case in- creased the percentage curing of a culture; but it did consistently broaden the range of concentrations capable of curing, thus allowing cured colonies to be detected at reasonable levels of acridine dye. All F+ strains which could be cured with acridine orange could also be cured with neutral red. Figure 6 summarizes the transfer and curing of the staining reaction. All strains which stained positively following conjugation with S. pullorum 35F".W6 or with staining re- action+ cultures derived from this strain both could be cured of the positive staining reaction and could transfer the permeability to eosin to an F- recipient. Low Frequency Transfer of Chromosomal Markers by S. +W Ellorum 35F 6--A fourth criterion of maleness of a bacterial strain is an ability to conduct chromosomal markers at a low frequency to a , , +W6 . , , reCipient culture. S_. pullorum 35F was tested for an ability to transfer chromosomal markers to E. coli 104. Following overnight incubation according to the shake r-flask method the washed mating mixture was spread on E-cys agar. This agar permits a natural contraselection of the donor in the presence of a high concentration of S. c_o_1_i_ cells, and a selection of thr+leu+thi+ recombinants. Two hundred-twenty recombinant colonies with the genetic constitution . - + + ,+ CYS thr leu thl arose. The sex factor contained by S. pullorum +W6 . . _ 35F 16 thus able to interact with the bacterial chromosome and to conduct chromosomal markers to a recipient strain. This sex factor, therefore, is classified as an episome. 48 000300 00000 000000Hm 0H3 0H00H00H 00000002 * .5300w 00 u OZ Hw0H000 00 n 02 H000H00H>00n3< 021000 , I oz A oz H. N oz 0380.0. .0 oz m m m m.o ms 06 md m.o *md ITIIIIII oz 0308 .Im - 0H00 Hm ooH ow C0 00 mm on 0H NH 0H m 0 N o HH0\m1 0H 000 H00H000 Ho 003000000000 .000 H000000 HHHHB 0000000 +.m Ho w0H00UII IN Hdmdflw 000 H000000 n m2 H0w000o 0000 00 u O< H000HH0H>00HHHH< .0oH00000 w0H0H000 05 .Ho w0H00o 000 0000008 .0 000th rmHu Hoo.m 03+ oH H y, 0, IN ME: (I 08 .mx _ 00000 00000 00000 _ mm+h 0000mm HH.HHH00HH0N .ImI HVOH+IIH Aw? mm rm 0? 9 H 00 .l 00 .l 4 .H m .H mm m2 O< _ O< mm+h 0000 mm H0000oHH0 rum 0 0? 0 0.5 A onfl .W mMIHHII .W , 0000mm H0000HH0mH .m.\ 00000 I 00000 0m .| 00000 03 HHoo .mm 0.3+.m mm HH %m 3.8 , mz H00.|A 0 Hoo.l/\ mmcHHHHIHOHme. 3+0 0: H m 00 3+0 3 H m J I. 0210 H .0 EH 08 .m 09:6 03 08 .m 7 +0 03 08 .m -0 30m E28050 .I0. 5.0 The frequency of chromosomal marker transfer by E. pullorum 35F+W6 to E. e_o_1_i_ 104 as calculated from Table X1 is Z. leO’6 recombinants/donor cell. This figure falls within the range expected of an F+ culture. The transfer of chromosomal markers by E. pullorum 35F+W6 to E. c_o_1i_ 104 is summarized in Figure 7. The ability of E. pullorum 35F+W6 to conduct chromosomal markers to an E. c_:_9_1_i_ recipient afforded an excellent opportunity to determine the relationship between acquisition and loss of a positive staining reaction and transfer and curing of a sex factor. Table XI presents the results of conjugating positively staining and cured cul- tures with strains of known mating types. The results presented in this table clarify the relationship between the staining reaction and donor ability of a culture. Those cultures which were assumed to be F+ on the basis of staining conducted chromosomal markers to ‘F- strains while the presumably cured strains did not form recombinants with F- recipients. Two of the horizontal columns in Table XI require explanation. When E. c_oli W6 was mated with E. _c_o_l_i_ 104F+35 a frequency of recombination five times greater than the value expected in an F+ x F cross was obtained. The higher frequency of recombinants in this cross is particularly surprising because of the nature of the markers transferred from E. (1111 104F+35. Strain 104F+3'5, having received only the sex factor from _S_. pullorum 35F+W6, remained genotypically thr. leu-thi-. Since a selection was made for thr+leu+thi+ recombinants, when E. (in 104F+35 transfers chromosomal markers to F- mutants in the E. c_cii W6 population the resulting recombinants are not ex- pressed. The fact that the number of recombinants is less than that observed in the W6 x 1.04 cross indicates that E. c_ol_i_ 104F+35 is acting as a donor producing nonviable recombinants. However, the fact that the number of recombinants is significantly greater than expected for Dono r + _S_. pullorum 35 F Recipient E. coli 104 Figure 7 . W6 51 \ E. coli 104 V Genotype + - + + + - - - F cys thr leu thi mot mal ind - + - - .- + +. + F cys thr leu thi mot mal 1nd - + + + + + cys thr leu thi mot mal ind Transfer of chromosomal markers from _S_. pullorum 35 F+ to E. coli 104. 52 TABLE XI. --Transfer of chromosomal markers by positively staining and cured cultures. * Mating Mating types No. recombinants/m1 E. coli x E. coli W6 x104 F+ x F- 250 W6 cured x 104 F' x F" o W6x104 F+35 F+xF+ 120 W6 x 104 F+35 cured F+ x F' 200 W6 cured x 104 F+35 cured F- x F- 0 W6 F+35 x104 F+ x F” 0 W6 F+104 x104 F+ x F” o E. pullorum x E. coli 35 x 104 F' x F' o 35 F+W6 x 104 F+ x F- 220 35 F+W6 x 104 cured F' x F' o =4: All matings were conducted according to the flask-shaker method. In all cases the donor was contraselected on m di devoid of an essential amino acid and selective for thr leu thi recombinants. 53 an F+ x F+ cross indicates that the F-factor is readily lost from strain 104F+35. This, as seen earlier, is the case. It may also be noted that E. (fli W6 cultures made F+ by receiv- ing a fertility factor which had been established in _S_. pullorum did not transfer chromosomal markers. Once again a host modification of the sex factor by passage through E. pullorum is indicated. E. pullorum 35F+W6 was classified as male strain of the basis of its positive staining reaction, its ability to transfer a sex factor, its ability to be cured with acridine dyes, and its ability to conduct chromosomal markers to an F- strain at a low frequency. Evidence of Donor Ability in S. pullorum 6 E. pullorum 6 fulfills all four criteria of 'maleness: (1) It pro- duces a strongly positive staining _. reaction, ~(2)can be dissociated from the staining reaction by treatment with acridine orange (see Table IX), (3) can transfer the ability to stain to E. gel} W6F- or to S_. pullorum 35 strr when conjugated by the tube mating method, (4) and conducts chromosomal markers to a second E. pullorum strain. There are two points of interest regarding the transfer and curing of a positive staining reaction in E. pullorum 6. First, when E. pullorum 35 strr was the recipient nearly'50% of the HZS+ strr exconjugant colonies stained positively, indicating an efficiency of transfer 16 times that observed when E. pullorum 35F+W6 acted as donor. Second, when the E. c_:_<_)l_i_ W6F- made staining reaction+ by mating with E. pullorum 6 was treated with acridine orange 100% curing at concen- trations from 16-3Zpg/m1 was affected. Thus, curing occurred at con- centrations of dye usually required for an E. 9211 strain but with a frequency characteristic of _S_. pullorum 6 (see Tables VIII and IX). 54 Repeated attempts were made to observe chromosomal transfer from E. pullorum 6 to an E. 9% recipient. Three strains, 104, AB 266, and AB 113, and three mating methods--the tube mating, flask- shaker, and gradient of transmission procedures--were used. In no instance were recombinants of any of the possible types selected found. E. pullorum 6 was capable, however, of conducting chromo- somal markers during an interstrain conjugation with _S_. pullorum 353trrhis-pro- ile-ara-mtl-gal-xyl- (s ee below). Evidence of Donor Ability in S. pullorum 35 strr F'lac+ strains + 6 Transfer of an F'lac _particle ELS. pullormn 355trr F'lac+. cultures--Forty E. pullorum 35strrlac+staining+isolates were tested qualitatively for the ability to transfer the lactose marker to a chloro- mycetin resistant mutant of E. pullorum 35 by conjugating the strains according to the tube survey method. After the mating period the conjugation mixtures were spotted on PR-lac, Cm(5iing/m1).agar. All forty cultures donated the lac+ marker at a high frequency, since all spots of the mating mixtures were confluently yellow. Control spots of the recipient strain showed no sign of fermentation. No growth of the donor strain was observed in donor control spots. The forty E. pullorum 35 Cmrlac+ cultures obtained from the previous mating were similarly tested for the ability to transfer the capacity to. ferment lactose to E. pullorum 355trr. The matings were carried out as above. .The donors were contraselected on PR-lac, str agar. All forty E. pullorum 35Cmr1ac+ cultures simultaneously donated the 1ac+ determinant and the ability to stain on EMO agar to the recipient strain. One of the initial E. pullorum 35 strrlac+ isolates was analyzed quantitatively for an ability to transfer the lactose marker. This 55 culture was conjugated with E. pullorum 35Cmr according to the tube mating method. At the end of the incubation period the mixture was diluted 106 and plated on Levines EMB-Cm agar. Lactose positive colonies were indistinguishable from lac- colonies on this medium. The colonies were transferred to Levine's EMB agar. Thirty-five percent of the Cmr colonies fermented lactose on Levine's EMB agar. One of the resulting lac+Cmr cultures was mated with E. pullorum 35strr and plated on Levine's EMB- str agar. Four-tenths percent of the resulting strr colonies were lac+. Two factors appear to be responsible for the low frequency of transfer. The lac+Cmr cultures consistently grow to a maximum cell count which is one-tenth that of _S_. pullorum 353trr; and 1ac+Cmr cultures appear to be less efficient in transferring episomic elements to recipient cultures. To compensate for the first of these factors the mating was repeated using a lO-fold concentrated culture of the Cmr donor. In this second cross 4. 6% of the recipient colonies received the lactose character from the donor. Efficiency of Transfer of an F‘lac+ plasmid from 3 + + s. pullorum 35F'lac to s. pullorum 358trr--E. pullorum 35F'lac and _S_. pullorum 353trr were mated in a ratio of 1:20 in a 125 m1 Erlenmeyer flask. Samples were withdrawn at various time intervals, diluted 105,“. and spread on the surface of Levine's EMB-str agar. After 48 hours incubation at 37C the plates were scored for the number lac+ colonies they contained; and the percent lac+ colonies was computed. The results of this mating are presented in Figure 8. The number of lac+ colonies increased to a maximum value of 85.2% within 25 minutes. This rapid transfer of the F'lac+ plasmid is characteristic of a cytoplasmic factor. Seventy percent of the recipient population received the F'lac+ factor within 17. 5 minutes of the onset of mating. This figure represents approximately three times the interval necessary for 70% transfer of ‘.n.--~—-¢ colonies + Pe rc ent lac 100- 56 A r 30 4'0 5'0 6?) 7‘0 8'0 90 160 11'0 12'0 Time (minutes) Figure 8. Efficiency of transfer of the F'lac+ plasmid from E. pullorum 35F'1ac+ to E. pullorum 353tr1'. 57 + . . an F factor in E. coli and suggests a longer time requirement for the formation of effective pairs in a E. pullorum intrastrain conjugation. Removal of the F'lac+ factor with ac ridine dyes--The original donor of the F'lac+ episome, E. QEAB785, spontaneously throws off 1% lac- segregants. The majority of these segregants retain the staining properties of a male culture. The lac- segregants are not due, then, to loss of the entire F'lac+ particle. Two possibilities as to the origin of the lac- segregants remain. The lac- colonies may arise from a spontaneous mutation of the lactose marker or from a mitotic recombination of the F' episome with the bacterial chromosome carry- ing a lac- gene, producing an F'lac- particle (Jacob and Wollman, 1961). Since the rate of segregation far exceeds the mutation rate of the lactose marker, the second possibility is the more likely. It is known that the presence of one promotor gene in a cell hinders the introduction of a homologous promotor into the same cell. A lac- segregant of E. 9911 AB785 receives an F'lac+ particle whose origin was E. coli AB785 from E. pullorum at a frequency of 0. 05% when the two strains are mated according to the tube mating method as compared to a frequency of 33% when an F- E. S2_l_i_ is substituted for the lac- segregant. Three criteria, then, determine whether the loss of an ability to ferment lactose in an F'lac+ strain, no matter if it occurs spon- taneously or following ac ridine treatment, is due to the loss of the F'lac+ or to recombination with the donor chromosome. If the F'lac particle is lost the culture will have lost the staining reaction and ability to ferment lactose and may be reinfected with the same F'- element. + The E. pullorum 358trrlac and the E. pullorum 35Cmrlac+ cultures which had been used for the quantitative analysis of the transfer 58 of the lactose character were treated with ac ridine dyes to determine their ability to be cured. The curing data for these strains are pre- sented in Table XII. Both cultures were simultaneously dissociated from the ability to ferment lactose and to stain on EMO agar by treatment with either ac ridine orange or neutral red. Seven cured E. pullorum 353trr1ac+ cultures were mated with a tenfold concentrated culture of E. pullorum 35Cmr by the tube survey method. Similarly fifteen cured E. pullorum 35Cmrlac+ isolates were mated with E. pullorum 353trr. The mating mixtures were spotted on PR-lac agar contraselective for the donor. All of the cured cultures were reinfected with the same F'lac+ particle they had lost and thus again became 1ac+ and staining reaction+. The transfer, curing, and superinfection data are summarized in Figure 9. Transfer of Chromosomal markers by strrF'lac+ Cultures of + S. pullorum--Forty E. pullorum 353trrF'lac cultures were tested for an ability to conduct chromosomal markers to E. (go—Ii 107. Thirty- nine of the cultures donated only the lactose marker. One exceptional culture donated the ability to ferment both lactose and galactose. Cells receiving these two markers gave a positive staining reaction and were able to transfer both carbohydrate markers and permeability to eosin to a second recipient. On treatment with either acridine orange or neutral red both sugar markers and the positive staining reaction were removed simultaneously. The cured E. 921.1. 107 F'laci-gal+ isolates were reinfected with the F'lac+gal+ particle from E. pullorum 358trrF'lac‘I-gal+ restoring their ability to ferment and to stain. Contraselection of the donor in the reinfection process was accomplished on MB— BCP agar. The spots of mating mixtures on MB-BCP agar were replicated to PR-gal and PR-lac agar to differentiate fermenting from nonfermenting cultures. 59 0000030 00000 00000000 05 00.000000 000000002 . * .000 #000000 n #02 “090000 0000000 u 04 20.260w on n OZ $000000 00 u OZ "0000000>00no.< H mmo.o H OZ mz A oz 0 +UdH7m HEUI. Cam 0 02 N A 02 0000o20m .m m2 0 OZ 0 m .m H H N . Alfie .0 OZ +0007m 00am... O< A 02 0 NH o0 u .. OH *m OZ 00000020m .m 0>0 000000 0.0 0000um 000 ow oo 00 Nm ON 00 NH. 00 w 0 N o 0000\m1 0m 0%0 000000 0.0 no 003000000000 .658: 83230 .m +6670 06 0:30-100“ 0005. 60 + E. coli AB785 F'lac E. pullorum 35 strrlac /-\ 4’. + E. ullorum 353tr rlac ACE E. pullorum 35Cm I.1ac+ E. ullo rum E. pullo rum 35Cmrlac - —353trrlaC' _. pullorum 35 Cmrlac+ r + AO E. pullorum 35str lac \ ( _ _. pullorum 35Cm rlac . . + . . Figure 9. Transfer and curing of F'lac particles in E. pullorum 35. Abbreviation: A0 = ac ridine orange. 61 It appears, then, that the majority of the lac+ E. pullorum iso- lates are F'lac+ and that one isolate carries an F'lac+gal+merogenote. Since none of these cultures transfer chromosomal markers, the sex factor5 they contain behave as plasmids rather than as episomes. It is not surprising that an F'lac+ particle is not able to integrate into the E. pullorum chromosome. The lac- marker in the Salmonellae because of its stability is considered to be the result of a deletion. In E. typhimurium all evidence indicates that the entire lactose region of the chromosome is missing (Zinder, 1960b). The mechanism of entrance of an F' particle into the chromosome requires pairing of the chromosomal marker carried on the F'merogenote with homologous region of the recipient chromosome. In the case of a Salmonella carry- ing a deletion in the lactose region the F' particle is unable to pair with the chromosome and remains cytoplasmic. It was hoped that the F'lac+gal+ would be able to integrate into the chromosome, since the addition of the gal marker to the plasmid introduces a region of homologous pairing between the exo- and endo- genote. However, repeated attempts to isolate Hfr mutants of the E. pullorum 35 strr F'lac+gal+ strain were unsuccessful. By plating E. pullorum 35 strr F'lac-*-gal+ culture in the stationary phase of growth on Levine's EMB agar it was possible to isolate approxi- mately 0. 05% Spontaneous variants of the F'lac+gal+ which were lac- staining reaction+. These variants on mating with E. 9311 AB266 according to the tube mating method transferred the staining reaction and the ability to ferment galactose simultaneously. On prolonged incubation of PR-lac agar these lac- variants fermented lactose. This ability identifies them as F'lac- cultures of Salmonella very similar to those described by Falkow and Baron (1962) in Salmonella typhosa. - + . . . . . These F'lac gal variants contain modified sex factors which probably arose from recombination with the donor chromosome. 62 These sex factors must contain either the lac- genes of E. pullorum, if they exist, or whatever gene(s) lie adjacent to the gal region in this organism. Thus, the F'lac+gal+ merogenote includes a large region homologous to the E. pullorum 35strr chromosome. It was anticipated that Hfr mutants might be isolated from this strain. Evidence of a Conjugation Process Two criteria determine whether genetic transfer is the result of a conjugation process. First, it must be shown cytologically that cell contact and subsequent steps in the conjugation process do occur; and, second, it must be shown through genetic evidence that cell contact must occur. Electron Microscopic Evidence of Conjugation between E. coli AB785 and S. pullorum 353trr--The conjugation process consists of three distinct steps: (1) formation of an effective conjugal pair, (2) conduction of genetic material from the donor to the recipient cell, and (3) formation of a recombinant cell. The first two steps may be evidenced by electron mic rosc0pic examination of mating mixtures. A. Formation of Effective Conjtgal Pairs--The formation of effective conjugal pairs proceeds in three stages: 1. Contact between Cells--Figure 10 shows an E. coli and an E. pullorum cell which have established contact. The cell walls of both cells are distinguishable at the point of contact. Contact was also observed to occur between two E. c__ol_i cells. Whether these con- tacts would evolve into effective pairs depends upon the mating types of the two cells in contact. In all likelihood the contact between two E. c_:_<_3_l_i cells would not have resulted in an effective pair, since both cells are of the donor type. However, any donor culture contains a small number of recipient cells, these having undergone a single mutation 63 Figure 10. Contact between an E. pullorum and an E. coli cell. 64 resulting in loss of the sex factor. Thus, two E. _(_:_9_l_i cells may in some cases form an effective pair. 2. Disappearance of Cell Boundaries--In order to observe the dissolution of cell walls between cells of a conjugal pair mating mixtures were subjected to negative staining by phosphotungstic acid (PTA). There are several advantages to PTA staining. The bacteria are fixed so as to allow air drying at room temperature with- out producing distortions of the cells or their relationship to one another. PTA fills in those areas of the cell which are empty, making these areas electron dense. Cytoplasmic characteristics and flagella are distinguishable, making them identifying markers. E. pullorum stained with PTA possesses neither flagella nor fimbrae. The cell wall is thin and difficult to distinguish. E. coli stained with PTA, on the other hand, possesses both flagella and fimbrae, and is approximately twice the size of the E. pullorum cell. Because of the several structural differences between E. ‘gcii and E. pullorum intrastrain conjugal pairs (E. c_o_l_i-E. (all) may be distinguished from intergeneric (E. pullorum-E. (lo—1i) mating pairs. Both in the case of the homologous E. ggl_i_-E. 93E conjugation and in the case of the heterologous E. gill-S_. pullorum matings (Figure 11) cell walls separating members of the pairs become indis- tinct in the area of contact. 3. Formation of a Cytoplasmic Bridge--In order to observe the cytoplasmic bridge connecting members of a conjugal pair mating mixtures fixed either by PTA staining or by exposure to the vapors of osmic acid were shadow casted with tungsten. In Figure 12 the bridge between members of an E. c_qE-E. pullorum pair is dis- tinguishable. The use of a high contrast photographic paper and a long exposure time to emphasize the area of the conjugal bridge has ob- scured flagellar detail. 65 Figure 11. Disappearance of cell boundaries betWeen members of an E. coli—E. pullorum conjugal pair. 66 Figure 12. Demonstration of a cytoplasmic bridge connecting members of an _Ei. coli-E. pullorum conjugal pair. 67 Shadow casting was found to be inferior to either PTA or uranyl acetate staining (see below) for demonstration of the conjugation process for the reason that the internal detail of the cells is obscured. After shadow casting conjugation can not definitely be distinguished from cell overlap or cell contact. The ability of PTA staining to de- tect contact has been indicated above. Figure 13 points out the ease with which cell overlap can be distinguished by UA staining... Uranyl acetate staining proved excellent for demonstrating not only the con- jugation bridge, but also the relationship of the donor to the recipient nuclea r appa ratus . B. Transfer of Genetic Material--The transfer of genetic material from a donor to a recipient bacterium via conjugation has not previously been observed cytologically (Clark and Adelberg, 1962). An attempt was made to demonstrate nuclear transfer by means of uranyl acetate (UA) staining. Uranyl acetate is a DNA specific stain. At longer contact times RNA and protein stain lightly (Smith and Melnick, 1962). Uranyl acetate fixes bacteria much as does PTA. Figure 14 demonstrates the diffuse nature of the DNA in E. pullorum. Figure 13 demonstrates the bipolar arrangement of the nuclear material in E. <_:_ol_i. Since neither fimbrae nor flagella are stained by uranyl acetate, the differences in nuclear aggregation and in size are the only reliable markers for distinguishing the two types of cells. Figures 15 and 16 demonstrate the transfer of genetic material (UA stained DNA) between members of an E. coli-E. pullorum conjugal pair. Figure 17 is of some interest in that it raises a pertinent question. Since both cells involved in the pairing are E. coli and they are 68 Figure 13. Overlapping cells of E. pullorum and E. C_O_1i ' stained with uranyl acetate. 69 Figure 14. Uranyl acetate stained E. pullorum. Figure 15. 70 Transfer of DNA between members of an E. coli-E. pullorum conjugal pair. 71 Figure 16. Transfer of DNA between members of an E. coli-E. pullorum conjugal pair. 72 Figure 17. E. coli cells connected by a DNA filled tube. 73 connected end to end by a DNA filled tube the question arises: does this relationship indicate genetic transfer via conjugation or me rely a stage in the division cycle of E. coli? A comparison of Figures 17 and 18 suggests that the association represents conjugation. In this figure the plasmodesma connecting two cells in the terminal stage of division may be seen clearly. The plasmodesma is devoid of UA stain- ing material. Figure 19 shows an E. coli cell at an earlier stage of division. The cell wall separating the two daughter cells had already been laid down at this stage. Thus, during terminal separation one would not expect the DNA of the daughter cells to be continuous but would, rather, expect the plasmodesma to be free of nuclear material. Figures 20 and 21 show E. SEE pairs stained with UA. In all cases a conjugation tube is visible. Genetic Evidence of Cell Contact A U-tube experiment was conducted to demonstrate that cell contact must occur between E. 321.1 AB785 and E. pullorum 355trr in order for genetic transfer to occur. The U—tube consisted of the chimneys of two small millipore filters separated by a millipore membrane (420 mu pore size). Five ml of the donor culture was added to one side of the apparatus and an equal volume of recipient to the other. The fluid from the donor culture was allowed to flow by gravity into the chamber holding the recipient culture. When nearly all of the liquid had drained from the upper section the apparatus was inverted. This process was repeated over a period of 24 hours. At the end of this time the recipient culture was diluted 10'6 and plated on Levine's EMB - str agar. No lac-'.strr colonies developed. A minimum of 200 lac+ recipients arose when the experiment was repeated without the millipore membrane separating the donor and recipient cultures and the same dilutions were performed. 74 Figure 18. Plasmodesma connecting E. coli cells in a terminal stage of division. 75 Figure 19. An E. coli cell in an early stage of division. 76 Figure 20. E. coli conjugal pair stained with uranyl acetate. 77 Figure 21. An E. coli mating pair stained with uranyl acetate. 78 Isolation of Hfr Strains from F+ and F' S. pullorum Cultures A UV-irradiated culture of E. pullorum 35F+W6 when replicated to E. c_cll_i_AB266 cells on E-cys agar gave rise to recombinants with the genetic constitution pro+thr+leu thi gal+mtl+cys-mot+ind+strr staining-. Areas of the donor giving rise to these recombinants were purified and identified as Hfr variants of the F+ culture. Four Hfr strains, Hfr-l-4, were isolated. Because the selection was the same in each attempt to isolate Hfr mutants all four gave recombinants of the same genetic constitution and are presumed to have the same origins. Strain Hfr-l was selected as donor for the genetic analysis of the gross structure of the E. pullorum 35 linkage group. An Hfr variant, Hfr-5, was similarly isolated from an E. pullorum 35F'lac-gal+culture. Selection in this case was accomplished on E-pro, cys, leu agar. It is interesting to note that the recombinants formed by mating E. pullorum Hfr-l with E. go_l_i_ AB266 contain over half of the genetic constitution of the donor strain. These were shown to be stable recom- binants by streaking on EMB-carbohydrate agars. When various Hfr strains of E. c_<_)_l_i_ were used as donor and _S_. pullorum 35 the recipient stable recombinants carrying a large portion of the donor markers were not formed. The reason for this unidirectional integration of donor determinants remains unexplained. Recipient Ability of S. pullorum 35 With the isolation of Hfr variants of male E. pullorum 35 strains the S. pullorum conjugation system was supplied with satisfactory donors. It remained to determine whether the entire E. pullorum 35 population would receive genetic material or whether a recipient variant must be isolated. 79 The recipient ability of E. pullorum 35 was tested as follows. Two matings were performed. E. £9_l_i_ AB785 was conjugated with E. pullorum 353trr and with E. pullorum 353trr E' lac+gal+ which had been cured of the F-factor by treatment with ac ridine dye. If E. pullorum 35 were an F0 culture similar to E. typhimurium, then the E. pullorum culture which had received an F'merogenote from E. c_o_l_i would repre- sent an F- mutant of the F0 population, since only an F- cell may receive genetic material. Thus, one would expect this selected F-, once cured of the sex factor it carried, to receive the F'lac+ factor from E. 3911 at a higher frequency than would the E. pullorum strr culture. The donor and recipient cultures were conjugated according to the tube mating method. Each mating mixture was diluted 10"5 and plated on Levine's EMB-str agar. When E. pullorum 35 strr F'lac+ gal+cured was the recipient strain, 1. 03% of the resulting colonies were lac+. When E. pullormn 358trr was the recipient, l. 03% of the surviv- ing colonies received the lac+ determinant from E. e_o_liAB785. E. pullorum 35, then, is an entirely F- population. Evidence of Intrastrain Conjugation The final consideration in the establishment of a conjugation system in E. pullorum was to determine whether a donor E. pullorum strain could conduct chromosomal markers to an F- _S_. pullorum culture. The advantage of an intrastrain conjugation system over an intergeneric one is that problems of integration resulting from chromo- some nonhomolo'gies .are eliminated. S_., pullorum 35F.“W6 was mated with _S_.. pullorum 35'strlihis-ara- xyl- according to the flask- shaker method. The mating mixture was spread + on W-cys,str agar to contraselect the donor. Eighty-eight his strr 80 colonies arose per plate. These recombinants had the genetic consti- . . + + - r . . tution his ara xyl str staining Conjugational Analysis of the S. pullorum Chromosome Three methods of genetic analysis were employed to determine the order of several genes on the E. pullorum 35Hfr-1 linkage group. These methods, mapping by time units, gradient of transmission, and genetic constitution of recombinants, were used interchangably. The result of each technique was the same--the ordering of the genetic determinants. Choice of a technique was governed by the frequency of recombination and the precise information sought from each mating. Reasons for the selection of a particular method of analysis will become apparent on analysis of the individual experiments. A recurrent problem during the conjugational analysis of the E. pullorum chromosome was the choice of a medium which would contraselect the donor and select for recombinants of a given type with- out decreasing the frequency of recombination. In attempting to over- come this problem several selective media and several techniques of selection were employed. Regardless of the selection procedure the order of markers determined was unaltered. Analjsis of the S. pullorum Linkage GrouLby Intergeneric Conjujation- -E. pullorum Hfr-1 was mated with E. coli AB113. At fif- teen minute intervals the mating pairs in one ml samples were inter- rupted by the syringe method. One-tenth ml aliquots of 10°, 10‘, 102 dilutions were plated on E-medium 1 without streptomycin to select for 1113+ cells. The donor was not contraselected. After 48 hours incubation at 37C one-hundred his+ colonies from each time sample were transferred to E-medium 1 without streptomycin and were repli- . . 1' . . cated to a medium selective for str cells. Thirty-eight percent of 81 the his+ colonies were strr. The frequency of genetic recombination in this cross was 61. 2 his+strr recombinants/100 Hfr cells. ' The genetic constitution of the his+strr colonies was determined by replicating to media selective for thr+leu+thi+, gal+, mtl+, and xyl+ recombinants and by transferring each colony to SIM agar. The inheri- tance of each donor marker among the his-I-strr recombinants was analyzed as a function of time. The results of the analysis of these recombinants are presented in Figure 22. Each marker begins to enter zygotes at a specific time after the onset of mating. The various markers can therefore be arranged in a sequence according to their times of penetration of recipient cells (Hayes, 1964; Jacob and Wollman, 1961). One feature of Figure 22 requires explanation. Since the leu- determinant is the first of the three nonselected markers to enter the recipient cell it is expected that this marker should appear most frequently among the his+strr recombinants. It appears, however, least frequently. This anomolous frequency results from the nature of the leucine marker in E. pullorum Hfr-1. The leucine marker in E. pullorum Hfr-l mutates at a high rate from an ability to an inability to synthesize the amino acid. Thus, only a portion of the Hfr p0pulation contributes the leu- determinant to the recipient population. All of the his+strr recombinants tested were ind-xyl-mtl- and gal-. These markers are not transferred at a high frequency to recipient cells. The absence of these markers in the his+strr recombi- nants defines the proximal segment of the E. pullorum Hfr-1 chromosome as that length of the linkage group carrying genes from the histidine through the cysteine marker. A map of the proximal segment of the E. pullorum chromosome as defined by the four markers tested is shown in Figure 23. 82 100. 90 ‘- 80-- 70.; ' ’ ’ mot” X X X cys- 604- ‘ A- leu 50 T ’ 7. + 40,. . 30 1- 20 .. 10.. ’ - \\ O a 4“ 4 l I _n l 1 I ‘ U I I I I l l 10 20‘ 30 40 50 60 70 80 90 100 110 120 Time (minutes) Figure 22. Positions of the mot, cys, and leu genes on the E. pullorum Hfr-1 linkage group as determined by intergeneric conjugation. 83 ~‘.W“N K C Y S \ leu Figure 23. Mapping of the proximal segment of the E. pullorum Hfr-1 linkagegroup in time units. 84 The rate of chromosomal transfer from E. pullorum Hfr-1 to E. c_gE 113 may be determined from the information presented in Figure 22, according to the equation: In p = -kt, where p = relative frequency of transfer of a marker, k = rate of chromosomal transfer, and t = time of entrance of the marker in minutes (Jacob and Wollman, 1961). The rate of chromosomal transfer in this experiment was 0. 022/min. If the rate of chromosomal transfer is constant during conduction of the proximal segment, then the frequencies or relative frequencies of marker transfer when plotted against time of entrance for each marker should form a straight line (Taylor and Adelberg, 1960). Figure 24 shows the results of such a graphic representation of the data. According to this graph the histidine determinant is expected to penetrate the recipient cell at approximately 5 minutes after the onset of mating. A second conjugation of E. pullorum Hfr-1 and E. coli AB113 was performed to determine the position of the his+ marker in time units. In this mating conjugal pairs were disrupted by means of a Waring blendor. The donor was contraselected on W-cys, str agar. Recombinant colonies required 7 days incubation at 37C to develop. Figure 25 shows the results of this cross. Figure 26 describes the alteration of colony forming units of the donor and of the recipient cultures with time‘in this mating mixture. This information is re- quired to compute the number of recombinants per 100 Hfr cells. The frequency of recombination in this cross was approximately 0. 005 of that observed when selection of his+ recombinants was per- formed on E-medium. Thus, W-imedium while effective in contra- selecting the donor in an E. pullorum-E. c__ol_i cross also contraselects a large percentage of the recombinant cells. Percent male marker 100. 90- 80d 70. 60' 50‘ 40' 304 20. 10; 85 l l I . 1 I I l i 1 T W l 10 20 30 40 50 60 7O 80 90 100 110 Time (minutes) Figure 24. Relationship between percent male marker transfer and time of entry of male markers. 120 .7‘ 86 - his+strr Time (minutes) Figure 25. Time of entrance of the his+ marker into E. coli AB113. 1x107 1x106- I 87 a L . x a Total cell count x X X Donor cell count 0 o 0 Recipient cell count I l L A l l I r l I T I 1‘ i I I 10 20 30 40 £0 60 70 80 90 100 110 120 l Time (minutes) . Figure 26. Growth curves of donor and recipient in an E. pullorum Hfr-l x E. coli AB113 mating mixture. 88 Analysis of the genetic constitution of the his+strr recombinants at 105 minutes revealed the following percent male markers among the recombinants: mot- 51.5, cys- 49.3, leu- 28.4. The figures for the cys- and mot- markers are comparable to those obtained in the previous c ros s . Kinetics of Effective Contact between S. pullorum Hfr-l and E. coli AB113--The kinetics of effective contact were studied by mating E. pullorum Hfr-1 with E. coli AB113, removing samples at specified time intervals, diluting gently 10-1, 10-2, 10-3, 10-4, and plating 0. 1 ml aliquotes on media selective for his+strr, gal+strr, xyl+strr, mtl+strr, gal+, xy1+, mtl+ recombinants (medial, 3, 4, 5, 6, 7, 8, respectively). The results of this conjugation are given in Table XIII. The curves obtained by plotting the number of recombinants as a function of time (Figure 27) indicate the rate of union formation for this particular population density. Each curve starts from the origin, indicating that effective contact formation begins as soon as the cultures are mixed. The curves rise to a plateau value at about 30 minutes. Thus, 9 after 30 minutes uninterrupted contact between the donor and recipient cultures no further specific pairs are formed. Location of Markers on the Distal Sefiment of the S. pullorum Hfr-l Linkage Grou --Figure 27 and Table XIII provide information on the location of markers of the distal segment of the Hfr-l chromosome. The plateau level of recombinants achieved for each selected marker is different. As seen earlier the final frequency with which a male marker penetrates the female cell is inversely proportional to its time of entry. Thus, the various plateau frequencies of transfer represent a gradient of transmission of these male markers to recombinant cells. 89 TABLE XIII. --Kinetics of union formation between E. pullorum Hfr-l and E. coli AB113. Time (min) No. recombina ts/ml his str lgal+strr xyl strr mtl strr Jal+ xy1+ mtl+ 5 3.8x106 5.3x104 0 3.6:.104 10 7.3x10" 2.9x10‘ 0 1.9x105 15 5.1x106 4.8x10‘ 0 1.9x105 30 6.3x106 7.1x104 0 2.6x105 45 2.0x107 7.81.104 0 1.8x105 60 4.4x106 17.6x104 0 2.6x105 75 2.81410" 1.0x105 10 3.2x105 90 2.3x10" 6.6x10‘ 60 2.6x105 105 4.3x10" 2.65.105 60 2.9x105 120 - 4.3x105 - 2.3x105 2.3x10" 1.1x1071.5x106 150 3.3x10" 4.9x105 40 2.6x106 3.1;.107 2.8x1074.8x105 180 4.73.107 1.05.10" 50 2.0x106 4.1x107 4.5x1071.3x106 90 0.60 69.2% o o o gal+strr x x x mt1+strr his‘istrr + or mtl strr recombinants/100 Hfr I‘ + No. of gal str l L 1 I I u I 1 I I 50 60 70 80 90 100 110 Time (minutes) Figure 27. Kinetics of union formation between E. pullorum Hfr-1 and E. coli AB113. 120 I I U1 .1 .5» +0 .0 O recombinants/100 Hfr O 1' . 0 str No. his 91 From Figure 27 it may be concluded that the mannitol marker precedes the galactose determinant into the recipient cell and that these two markers lie within a short distance of one another on the linkage group but are at a great distance from the histidine marker. The low frequency of transfer of the gal, mtl, and xyl markers (see Table XIII) places them on the distal segment of the chromosome. According to Figure 24 these male markers which have not penetrated the his+strr recombinants lie at a distance measured in time units beyond 55 minutes on the donor chromosome. It should be noted that in Figure 27 after about 90 minutes the number of recombinants of all types in a mating mixture increases sharply and proportionally. This rise is the result of the rapid division of the recombinants in the absence of a selective force. Table XIII provides one more indication of the order of genes on the distal segment of the E. pullorum Hfr-l linkage group. By com- paring the frequencies of the recombinant classes gal+strr : gal+, xyl+strr : xyl+, and mtl+strr : mtl+ it may be seen that of the three carbohydrate markers entrance of the mannitol determinant is least effected by selection for strr recombinants. The frequency of entrance of the xyl+ marker is most effected and that of the gal+ determinant is intermediate. It appears, then, that the gal and xyl markers are more closely linked to str than is mtl and that xyl is the closer of the two markers to the str locus. It is not possible to determine from these data, however, whether the precise order of these genes on the distal segment is mtl. . . . gal. . .xyl. str or mtl. . . . gal. . . str.xyl. The relative positions of the three carbohydrate markers were confirmed by analysis of the genetic constitution of the gal+strr and + the mtl strr recombinants (Table XIV). According to these data the gal and mtl markers lie close to one another on the linkage group 92 . . . + r + r , TABLE XIV. --Genetic constitution of gal str and mtl str recombinants. Selected markers Percent nonselected marke rs + r + r + r gal str mtl str xyl str + gal strr 100 100 39 + mtl strr 99 100 0 93 and the gal determinant lies closer to the xyl region than does the mtl marker. The order of several genes on the _S_. pullorum Hfr-1 linkage group as determined by intergeneric conjugation is summarized in Figure 28. Conjugational Analysis of the S. pullorum Chromosome by Intragaecies Corgjugation An Intrastrain Conjggation with S. pullorum Hfr-l-- E. pullorum Hfr-1 and E. pullorum 355trrhis-pro-ile-ara-mtl‘gal- were mated according to the gradient of transmission method, diluted 10‘Z ', 103 , and 10“ , and plated on W-ll and W-12 media. After seven days incubation at 37C the plates were scored for the number of colonies they contained; and the number of recombinants/ml and the frequency of recombination for each character were computed (Table XV). The frequencies of recombination indicate that the entrance of the proline determinant into a recipient cell precedes that of the iso- leucine marker. The frequency of recombination for the pro+ marker is comparable to that of the his+ marker in an E. pullorum Hfr-l x E. 32E AB113 cross. Thus, Hfr-1 behaves as a high frequency recombination donor in an intrastrain as well as in an intergeneric mating. The genetic constitutions of 100 pro+ and 100 ile+ recombinants were determined by replicating to E-media or PR-carbohydrate media selective or differential for each of the donor characters. The results of this analysis are given in Table XVI. Several points become evident on examining Table XVI. First, a true gradient of transmission of donor markers among the recombi- nants was not obtained, since donor markers on widely separated areas of the chromosome are transferred with the same frequency. The existence of a gradient of transmission of donor characters depends upon the random rupture of the linkage group during its conduction to 94 -leu Figure 28. The order of several genes on the E. pullorum Hfr-l linkage group. The exact distances between markers in parentheses was not determined. 95 TABLE XV. --Transmission of markers from E. pullorum Hfr-l to E. pullo rum 35 strrhiS'pIO'ile'ara‘mtl'gal" . Selected No. recombinants/ml No. recombinants/100 Hfr markers + r 4 pro str 1.03x10 0.212 + ile strr 3.33.103 0.07 TABLE XVI. --Genetic constitution of recombinants in an Hfr-l x _S. pullorum 353trrhis'pro‘ile’ara'mtl'gal" cross. Selected Percent male marker , + + . + + + + -s , , markers his pro ile ara mtl gal str staining + pro strr 100 100 100 100 100 100 98 14 , + r ile str 100 100 100 100 100 100 64 16 96 a recipient cell. In the event that the probability of breakage of the conjugal bridge and chromosome is low, recipient cells continue to receive donor markers until some experimental event such as plating on minimal-streptomycin agar (Jacob and Wollman, 1961) separates the mating pairs. Stable effective pairs in E. (3213 have been described by Taylor (1961) as being characteristic of very high frequency (Vhf) male strains. These donor cultures transmit terminal markers with a frequency from 20-200 times higher than the frequency of transmission .by ordinary males. Comparing the relative frequency of transfer of the gal+ marker from Hfr-1 to E. c_o_EABll3, on the one hand, and to E. pullorum 35 strrhis-pro-ile-ara-mtl-gal-, on the other hand, it is evident that the transfer to E. pullorum exceeds that to E. _c_0_11_ by a factor of greater than 100. The same increased frequency of transmission of markers on the distal segment of the linkage group in an E. pullorum intrastrain cross was found for the mannitol and streptomycin markers. Similarly, the pro+ and ile+ characters occurred more frequently among the recombinants than was expected on the basis of an Hfr-l x E. C3311 AB113 cross. The frequency of recombination for an early marker (e.g. pro+) was not increased, however, above the value of an early marker (e.g. his+) in an Hfr-1 x AB113 mating. Thus, it is unlikely that the high values for transfer of late markers is due to an increased ability of the donor and recipient cells to mate but must, rather, be the result of a characteristic of the conjugal pairs once they have formed. It appears, then, that E. pullorum-E. pullorum effective pairs are more resistant to disruption than are E. pullorum-E. c_fl pairs and that the formation of exceptionally stable mating pairs depends not only upon the characteristics of the donor but also upon the nature of the recipient strain. 97 A second point of interest evidenced in Table XVI is the occur- rence of a positive staining reaction among recombinants formed in an Hfr-1 xE. pullorum 35strrhis-pro-ile-ara-mtl-gal- cross. Sixteen percent of the ile+recombinants and 14% of the pro+ recombinants gave a positive staining reaction on EMO agar. The ability of these recombi- nants to stain indicates that they have received promotor genes from the donor strain. All of the staining+pro+ recombinants were strs, making them indistinguishable from donor cells. All of the staining+ile colonies, however, retained their streptomycin resistance. In this second case the staining+ile+ recombinants may have received the terminal Hfr gene from the donor and have excluded the strS region of the male linkage group by a recombination event during the formation of the recombinant chromosome. The question of whether the sex factor is always attached to the terminal end of the chromosome or sometimes splits into two parts, one distal and one proximal, both of which are necessary for fertility has not been decided (Jacob and Wollman, 1961). If the second mechanism of integration is possible, the staining+ile+strr recombinants may represent cells which have received only a portion of a donor chromosome containing a split sex factor. A third fact brought out in Table XVI is a shortcoming of W-media. Since 98% of the pro+ and 64% of the ile+ recombinants are sensitive to streptomycin, the W-media are not selective for E. pullorum strr cells. The fact that strS recombinants survive on W-media is probably the result of the long incubation time. After 7 days incubation at 37C most, if not all, of the initial streptomycin is inactivated. However, since all of the ile+strS recombinants do not stain on EMO agar, these media do appear to contraselect the donor cells and allow only recombinant colonie s to survive . 98 An Interstrain Conjugation with S. pullorum 6--E. pullorum 6 andE. pullorum 353trrhis-ile-ara-mtl-gal-xyl- were mated for 90 minutes. The conjugation mixture was diluted 10-2 and 10.3 and plated on media selective for ile+strr (W-ll) and his+strr (W-lO) recombinants. After 7 days incubation at 37C the plates were scored for the number of recombinants they contained; and the number of recombinants/ml and the frequency of recombination for each marker were computed (Table XVII). The recombination frequency for the ile+ marker is comparable to that obtained for both an E. pullorum Hfr-l x E. c1)_l_iABll3 and an Hfr-1 x E. pullorum 35strrhis-pro-ile-ara-mtl-gal- cross. E. pullorum 6, therefore, is classified as an Hfr male. The frequencies of recombination presented in Table XVII indicate that the ile+ marker lies closer to the origin of the donor chromosome than does the his+ marker. The genetic constitution of 50 ile+ recombinants is given in Table XVIII. Using the same arguments that applied to the Hfr-1 x E. pullorum 35 strr recipient cross it may be concluded that the conjugal pairs formed between E. pullorum 6 and a second E. pullorum strain are unusually resistant to disruption. Also, as in the previous mating, all of the staining+ile+ recombinants were strr and were also xyl... The same question as to the position(s) of the staining determinant may be posed in this cross. One point brought out by this cross but not by the previous intra- strain mating is the relationship between the xyl and str markers. In 96% of the cases whenever a recombinant was strr it was also xyl-; when the recombinants was strS it was also xyl+. The low frequency with which these two donor characters separate indicates that they lie within a short distance of one another on the E. pullorum 6 chromosome. This finding confirms the similar conclusion drawn from an E. pullorum Hfr-1x E. coli AB113 cross. 99 TABLE XVII. --STransmission of markers from E. pullorum 6 to p.ullorurn 35$t1‘rhIS' ile" ara" mtl gal xyl'. Selected No. recombinants/ml No. recombinants/100 Hfr markers ile+strr 5.0x103 0.43 + his strr 5.0x10Z 0.043 TABLE XVIII. --Genetic constitution of recombinants in an E. pullorum 6 E. pullorum 35strrhis"ile'ara'mtl'gal’xyl‘ cross. Percent male marker Selected + . + + + + + . s . . markers his ile ara mtl gal xyl str staining fle+strr 96 100 100 100 100 78 79 o 100 + Judging from the genetic constitution of the ile recombinants a reasonable order of entry of markers on the E. pullorum 6 chromo- some is indicated in Figure 29 below. O---gal---mtl---ile---ara---his---xyl---str Figure 29. The probable order of entry of markers on the E. pullorum 6 linkage group. Abbreviations: O - origin; gal = galactose; mtl = mannitol; ile = isoleucine; ara = arabinose; his = histidine. DISCUSSION The establishment of a conjugation system in a species of bacteria which has not previously been shown to be capable of mating depends upon (1) the identification or establishment of a donor culture and (2) the selection of a competent recipient strain. In the event that the conjugation system is to be used for a gross structure analysis of the bacterial chromosome, high frequency recombination (Hfr) mutants of the donor type must be isolated. Each of these Hfr strains should have a different point of origin on the bacterial chromosome so that all markers studied are transferred on the proximal segment of an Hfr chromosome. It'is this segment which is conducted to recipient cells at a high frequency and at a constant rate. These characteristics of transfer permit differences in frequencies of recombination for the proximal markers to be readily and accurately translated into time units, representing quantitative expressions of the amount of DNA separating the various genetic determinants. Early attempts to demonstrate sexuality in the Salmonella were unsuccessful (Zinder and Lederberg, 1952). Later several Salmonella species were found to be receptive to varying degrees to sex-factors of E. coli (M'akel'a e_t a_._1. , 1962). These male Salmonella strains would transfer their newly acquired fertility factors to other Salmonella and, with a greater efficiency, back to E. coli. In all cases the Salmonella strains were male only after mating with a male culture of E. coli under experimental conditions. For this reason the Salmonella have been considered to be universally female strains; and male strains of E. coli K 12 have been referred to as the sole source of maleness in 101 102 + bacteria (Jacob and Wollman, 1961). More recently an F'lac strain of Salmonella typhosa has been isolated from a natural habitat (Falkow and Baron, 1962). This culture contained the same lac+ genes (as did an F'lac+ strain of E. c_cfi and is presumed to have derived from that strain by means of an i_n v_iLo_ conjugation. A survey of forty-nine E. pullorum strains for maleness revealed one male strain, E. pullorum 6. This culture efficiently transferred its maleness and its chromosome to another E. pullorum strain but transferred its sex factor at a low frequency and chromosomal markers not at all to various E. _cEli recipients. If E. pullorum 6 has received a fertility factor from a derivative of E. 9.9.1.1 K12 through an i_n v32 mating, then the sex factor has become modified so as to be restrictive with regard to the nature of the recipient cultures with which it will effectively conjugate under the experimental conditions used. The concept of host modification of a sex factor has previously been evoked to explain the stability of an F'lac+ factor in E. abony + and the ability.of only certain F Salmonella to mate effectively with other Salmonella strains (Makelfi gt a_l. , 1962). + In this study the stability of F'lac E. pullorum cultures, the +W intensified staining reaction of E. pullorum 35F 6 and the increased + stability of an F strain of E. coli 104 following numerous passages, + the inability of E. coli W6 F strains derived from male cultures of E. pullorum to mate with E. coli recipients may be readily explained as representations of host modifications on F-factors. Like most Salmonella strains (M'sIkel'a e_t 11° , 1962) E. pullorum 35 + + readily received F and F'lac sex factors from E. coli donors. The + F culture of _S_ pullorum 35 fulfilled all four criteria of maleness. + The F'lac strains fulfilled the criteria of a male strain containing a plasmid sex factor. 103 Several Hfr strains suitable for the analysis of the linkage group + have been isolated from F strains ofE. typhimurium (Demerec and Sanderson, 1964), and both F+ and F'lac+ cultures of E. £32111 have given rise to Hfr strains (M'alkela, 1962). It was hoped, therefore, that the F+ E. pullorum 35 culture would yield Hfr strains with various orders of entry and that the F'lac+ E. pullorum culture would permit the isolation of an Hfr mutant with a predictable origin. An Hfr mutant of E. pullorum 35F+W6(Hfr-l) with the order of entry O--his--leu--mot--—-cys--mtl--gal--(xyl)--(st-r) was isolated. This strain behaved as a classic Hfr in that (1) it transferred a proximal segment of its linkage group with high frequency and a distal segment with low frequency to an E. c_o_l_i recipient. This Hfr strain was able to form exceptionally stable effective pairs with an E. pullorum 35 recipient and to transfer a large segment of its chromosome at a high frequency during intrastrain conjugation. (2) Hfr-l conducted donor markers in an ordered and sequential manner and (3) transferred the attached sex factor at a low frequency to recipient cultures. No Hfr mutants could be derived from any of the many E. pullorum 35F'lac+ isolates. These cultures were incapable of conducting chromo- somal markers. In this respect they resembled an F'lac+ strain of _S_. typhosa (Falkow and Baron, 1962) rather than an F'lac+ strain of E. abfly (Méikeléi e_t 11' , 1962). An Hfr culture was, however, derived from an _E. pullorum 355trr F'lac-gal+ strain. The origin and order of penetration of markers on this donor chromosome was not determined. E. pullorum 6 was found to be an Hfr strain with the probable order of entry O--gal--mtl--ile--ara--his--xyl--str. This Hfr strain also formed very stable effective pairs with an E. pullorum recipient. Since _E. pullorum 6 transferred the gal+ marker with a high frequency and the his+marker with a lower frequency whereas the E. pullorum Hfr-l donor conducted the his+ marker on the proximal 104 Segment and the gal+ marker on the distal segment of the chromosome, the E. pullorum linkage group must be a closed continuous structure in the F- and presumably also in the F+ cell and discontinuous during transfer from an Hfr mutant. Such a circular map conforms with the pictorial representations of both the E. 9.9.13. (Hayes, 1964; Jacob and Wollman, 1961) and of E. typhimurium (Sanderson and Demerec, 1964) chromosomes. One of the female strains of E. pullorum, strain 35, was tested for its recipient ability and was found to be a population of entirely receptive cells (F-) rather than a mainly nonreceptive F° culture as is E. typhimurium (Baron et a1. , 1959). Thus, in its recipient ability E. pullorum 35 closely resembles E. typhosa (Falkow and Baron, 1962) and E. M(Makela e_t 11' , 1962). Following the isolation of two E. pullorum donors and the identi- fication of a suitable recipient population the order of several genes on the E. pullorum donor chromosome was determined to be his--1eu-- mot--cys--mtl—-gal--str—-xyl. The exact positions of the pro and ile markers were not determined; however, it was established that pro lies closer to his than does ile on the Hfr-1 linkage group. The positions of the gal and str markers on the E. pullorum donor chromosome do not coincide with the location of these determinants on the E. c_0li_ and E. typhimurium chromosomes (Jacob and Wollman, 1961; Sanderson and Demerec, 1964). A composite picture of the order of genes on the E. pullorum chromosome derived from this study is presented in Figure 30. 105 (ile) cys mot (mtl) (gal) LE. pullorum 6 0 leu (str) (xyl) 10 min. ( )* ro Hfr-l p his Figure 30. A composite picture of the circular E. pullorum linkage group. Abbreviations: 0-origin; his=histidine; pro=proline; leu=leucine; mot=motility; cys=cysteine; ile=isoleucine; mtl=mannitol; gal=galactose; xyl=xylose; str=streptomycin. * The exact position in time units of the markers in parentheses has not been determined. SUMMARY Of forty-nine strains of E. pullorum surveyed for maleness one strain, E. pullorum 6, was male. This strain was found to be an Hfr culture and to conduct chromosomal markers to a second E. pullorum strain but not to an E. (BE recipient. One of the female E. pullorum strains was analyzed for its recipient ability both cytologically and genetically. The recipient ability of E. pullorum 35 was genetically characteristic of an F- rather than an F0 population. Electromicroscopic studies of E. QE- E. pullorum mating pairs demonstrated the ability of strain 35 to form effective pairs with and to receive genetic material from an E. coli donor. E. pullorum 35 readily received both an F+, F'lac+, and an F'lac+gal+ factor from E. 2913 male strains. The F+ and a lac- segregant of the F'lac‘lgal+ E. pullorum 35 cultures fulfilled all four criteria of maleness in bacteria. The F'lac+ isolates exhibited the characteristics of male strains containing a plasmid sex factor. 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