Received for publication 17 August F' factor once transfer had occurred. Those Lac7. by the sterile class of F' bacteria are also presented.

Transcription

1 PROPERTIES OF F' STRAINS OF ESCHERICHIA COLI SUPERINFECTED WITH F-LACTOSE AND F-GALATOSE EPISOMES HARRISON ECHOLS Department of Biochemistry, University of Wisconsin, Madison, Wisconsin ABSTRACT ECHOLS, HARRISON (University of Wisconsin, Madison). Properties of F' strains of Escherichia coli superinfected with F-lactose and F-galactose el)isomes. J. Bacteriol. 85: A study of F' superinfection of F' strains of Escherichia coli by F Lac P and F Gal indicates that superinfecting F Lac P may achieve a stable existence along with native F Lac P in approximately 1 % of the recipient F' population. The stable presence of F Gal, on the other hand, leads to a displacement or suppression of F Lac P when F Gal is used as a superinfecting agent or as the native episome. The majority of F' bacteria used as recipients neither acquire in stable form the superinfecting F-linked genes nor demonstrate gene activity immediately after attempted transfer, as judged by alkaline phosphatase synthesis directed by an F-transferred P+ gene. This failure to show gene activity suggests that the F' bacteria which are sterile as recipients exclude transfer rather than inhibit subsequent multiplication. F' and F' strains of Escherichia coli transfer the F-factor with high frequency to F- strains. F+- bacteria also transfer chromosomal genes with low frequency to F- (Lederberg, Cavalli, and Lederberg, 1952; Hayes, 1953); F' bacteria transfer certain extrachromosomal (F-linked) genes with high frequency to F- (Jacob and Adelberg, 1959; Hirota, 1959). F+ or F' by F+ crosses exhibit a much lower fertility than F+ or F' by F- crosses, as determined by reduced transfer of genetic markers from the F+ or F' used as donor (Hayes, 1953; Scaife and Gross, 1962). The present communication describes some experiments concerned with the nature of this reduced fertility, using the extrachromosomal genes transferred with the F factor by F' strains to follow the fate of the superinfecting F' episome (e.g., F Lac). Received for publication 17 August 1962 The fraction of F+ (Lac-) or F' (Lac-) cells which do not give Lac+ clones when mated with F' cells carrying F Lac+ (the majority sterile class) may have either excluded transfer or prevented multiplication of the superinfecting F' factor once transfer had occurred. Those Lac7 cells which yield Lac+ clones (the minority fertile class) may have been F- variants and added F Lac+, may have added F Lac+ as an "extra" F' factor, or may have excluded the superinfecting F' factor in a process producing Lac+ chromosomal (or episomal) recombination. This paper is concerned primarily with the possibilities described above in connection with the class of F' bacteria fertile as recipients. Some experiments which suggest the nature of the exclusion by the sterile class of F' bacteria are also presented. The interpretation of the fate of the superinfecting gene in terms of the fate of the superinfecting F' episome is based upon the assumption that the F factor is linked to the genes transferred with high frequency by F' strains. This assumption of F-linked genes appears plausible on the basis of the work of Jacob and Adelberg (1959) and Adelberg and Burns (1960) and will be used in this paper without further comment. It should be noted, however, that the experiments follow directly only the superinfecting gene and not the F factor. MATERIALS AND AIETHODS Nomenclature. F+-Male (genetic donor) type of E. coli K-12 which transfers F ("fertility factor") with high efficiency to F- female (genetic recipient) types and transfers chromosomal genes with low efficiency to F- types (Hayes, 1953; Cavalli, Lederberg, and Lederberg, 1953). F'-Ft variant diploid for a small genetic region which transfers these "extra" genes at a frequency corresponding to F transfer. The genes transferred at high frequency are believed to be linked to the F factor (Jacob and Adelberg, 262

2 VO)L. 85, 1963 SUPERINFECTION OF F' E.COLI ). F and F' are examples of "episomes" (Jacob and Wollman, 1958). Hfr-F+ variant transferring the bacterial chromosome with high efficiency (Hayes, 1953). Lac+ and Lac-; Gal+ and Gal-genetic markers for ability or inability to utilize lactose or galactose as energy source. TL+ and TL-; M+ and 1JT-genetic markers for ability or inability to grow without threonine and leucine or methionine. P+ and P-genetic markers for ability or inability to synthesize alkaline phosphatase. S8 and Srl-genetic markers for sensitivity or resistance to streptomycin. F Lac+-denotes F' strain able to exhibit F-linked (high frequency) transfer of lactose utilization. F Lac+/Lac-M---denotes F' strain able to exhibit F-linked transfer of the Lae+ marker and carrying the Lac- and M- markers on its chromosome. Strains. Only genetic markers pertinent to the experiments are listed. WV3747-F' isolated by Y. Hirota. F Lac+P+/ Lac+P+S8. W4520-F' isolated by Y. Hirota. F Gal+/ Gal+SS. KIO-Hfr S8. A derivative of the Hfr Cavalli which transfers P+ early. F'4-F Lac-P+/Lac-P+M-. Homogenote F' isolated from the heterogenote F Lac+P+/ Lac-P+M- containing the F Lac+P+ of W3747. F' 26-F Lac+P-/Lac+P-M-. Homogenote F' isolated by A. Garen from a heterogenate containing the F Lac+P+ of W3747. F 3-Lacp-SrF. A derivative of the Wl F- (Lederberg). F 15 Lac-P-R2-SrF-. Another derivative of the WI F-. B 8-Lac-Gal-M-SrF- (Morse no. 550). W3102-Gal-< (Lederberg). I'w'4/F3-F Lac-P+/Lac-PTL-. Derived from a cross of F'4 by F3. TV3747/B8-F Lac+/Lac-Ga1-M-. Derived from a cross of W3747 by B /B8-F Gal+/Lac-Gal-M-. Derived from a cross of W4520 by B8. F'26/F15-F Lac+P/Lac-P-R2-Sr. Derived from a cross of F'26 by F15. M1edia. Bacteria were grown in Penassay Broth (Difco). Selective plating was carried out on minimal S agar (Echols et al., 1961) containing 0.3% lactose (T Lac Agar), glucose (TG Agar), or galactose (T Gal Agar). Growthlimiting phosphate (6.4 X 10- M KH2PO4) was used in the T Lac and TG plates to create conditions of alkaline phosphate induction. The T Gal plates contained excess phosphate (6.4 X 10-4 M KH2PO4). Amino acids were supplied where needed at a concentration of 20 mg/liter. Streptomycin was used at a concentration of 100 mg/liter. EMB lactose and galactose agar was prepared as described by Lederberg (1947). Mating conditions. Penassay Broth cultures were mixed to give approximately 2 X 108 cells/ ml of the donor strain and 2 X 107 cells/ml of the recipient. The mating cultures were shaken at slow speed for 2 hr at 37 C and then plated on the appropriate solid media. Donor cells were used in the exponential phase of growth. F' by F- crosses were done routinely with exponential phase F- cells. F' by F' crosses were done using F' recipient cells from an aerated, saturated overnight culture. This procedure increased the number of F' recipient cells fertile to stable genetic transfer approximately 1,000-fold over that obtained using exponential phase F' cells as recipients (see Hayes, 1953; Taylor and Adelberg, 1961). The transfer frequency in F' by F- crosses using a saturated F- culture was unaffected or sometimes slightly lower than that observed using an exponential-phase F- culture. In the crosses in which phosphatase activity was followed, standard F' by F' cross conditions were employed for both F' and F- recipients. Alkaline phosphatase assay. Alkaline phosphatase activity was assayed on limiting phosphate plates by spraying with a 15 mg/ml solution of p-nitrophenylphosphate (NPP) in ph 8.0, 1 M tris(hydroxymethyl)aminomethane (tris) buffer (Garen, 1960). P+ colonies turn yellow as the dephosphorylated p-nitrophenol is liberated. Quantitative assays on liquid cultures were performed as previously described (Echols et al., 1961) by following the rate of change of optical density at 410 m,u produced by p-nitrophenol liberation. Samples were prepared for assay by centrifuging, washing by centrifugation with 0.1 M tris buffer (ph 7.4), resuspending in 0.1 M tris buffer (ph 7.4), and shaking with a drop of toluene for 40 min.

3 264 ECHOLS J. BACTERIOL. RESULTS Strains containing native and superinfected F Lac P. An example of the attempted addition of a superinfecting F' factor of derivation similar to the original or native F' factor was first studied. For this purpose the F Lac P episome from strain W3747 was used as F Lac-P+ and F Lac+P- so that the native and superinfecting F' factors could be distinguished. (The closely linked markers Lac and P exhibit typically high frequency F' transfer by the F Lac+Pt WV3747 strain; see Table 1.) A strain carrying the chromosomal markers Lac- and P- and episomal F Lac-P+ was used as the Ft recipient. This F Lac-P+/Lac-P- (and TL-) strain was crossed with a strain carrying episomal F Lac+P-, and Lact clones were selected on minimal plates containing lactose as the energy source and growth-limiting inorganic phosphate to induce alkaline phosphatase formation in P+ cells (T Lac Agar). The F Lac+Pparent was eliminated by its methionine requirement. The Lac+ colonies were scored for P, and Lac+P+ presumptive superinfected (S) strains were purified by streaking once on minimal lactose plates and again on EMB lactose plates. The transfer frequencies for P and Lac in the superinfection experiment are given in Table 1 along with the frequencies for a cross of the same F Lac+PT strain with a Lac-PF- (the F- from which the F' recipient was derived). The frequency of Lac+ transfer is drastically reduced in the F' by F' cross, but the number of Lac+ colonies which possess the P+ marker of the native F LacP+ is substantial. This frequent occurrence of P+ and Lac+ together suggests the addition of the superinfecting F' factor as an additional episome. To test for the presence of both F' factors, five S strains with the presumed genetic structure F Lac+lT/F Lac-P+/LacP- (and TL-) were tested for high frequency transfer of Lac+ and P+. The S strains were crossed with a Lac-P-F-; Lac+ transfer was determined by plating on minimal lactose plates, and P+ transfer by plating on minimal glucose plates and scoring for P+. The S parent was eliminated by its TL requirement. Four of the five strains transferred Lac and P to the Ft with the very high frequency (greater than 80%) characteristic of F' by F- crosses and one transferred Lac and not P. The four S strains TABLE 1. Transfer of P and Lac in F' by F' and F' by F- crosses* DLac epdisnore Recipient genotype Lac+ recipients episome transfer ~~~~also P+ % % F Lac+P F Lac-P+/Lac-P t F Lac+tP Lac-P-F- 90 F Lac+P+ Lac-P-F * Crosses were carried out under standard mating conditions, and mated cultures were plated on T Lac Agar. The donor episomal strain was eliminated by its methionine requirement. Lac+ colonies were scored for P+ by spraying with NPP. The Lac+P- donor was strain F'26; the Lac+P+ donor W3747; the F' recipient F'4/F3; and the F- recipient F3 (see Materials and Methods). t There exists partial complementation between the P- mutation of the donor F' and the P- mutation of the recipient which made scoring for P+ by NPP spray subject to possible error because of some yellow color in colonies containing cells with only the two P- mutations. These experiments were repeated with a noncomplementing pair of P- mutations without change in the numerical results. In testing for P+ transfer by the S strains, a noncomplementing P- recipient was used. The complementation serves the positive function of showing that the donor F' does actually transfer the P region. transferring both Lac and P with high frequency thus exhibited the characteristics of F' transfer for both native Pt and superinfecting Lac+ markers. The segregation behavior of these high frequency transfer S strains also tended to support the interpretation that the superinfecting F' episome had been added. F' bacteria segregate cells lacking the F-linked marker (Jacob and Adelberg, 1959). If the S bacteria carried both F Lac+P and F Lac-P+, segregation events involving either one or both F factors might be expected to occur. Single segregation events should produce Lac-P+ and Lac+P- phenotypes; double segregation events LacP-T (the chromosomal markers). If the S bacteria carried Lac+ and P+ as a single F Lac+P+, segregation events should produce predominantly Lac-P- phenotypes. The results of the segregation analysis are summarized in Table 2. The high frequency of singly defective segregant phenotypes (Lac+Pand Lac-P+) argues for the presence of both

4 VOL. 85, 1963 SUPERINFECTION OF F' E. COLI 265 TABLE 2. Segregation pattern of F Lac P superinfected strains* P- seg- Lac- Presumed genotype regants also seg- regants Lac' also P- % % F Lac+P-/F Lac-P+/Lac-P- (1) F Lac+P-/F Lac-P+/Lac-P- (2) F Lac+P+/Lac-P- 4 4 * Cultures were grown to approximately 2 X 108 cells/ml and then diluted and plated on minimal glucose agar (TG Agar) to score for P- segregation (by NPP spray) and on EMB Lactose Agar to score for Lac- segregation. Approximately 1% segregant colonies were found. Twenty-four P- colonies were picked and streaked on EMB Lactose Agar and TG Agar to score for Lac character and check P. Twenty-four Lac- colonies were also streaked on TG Agar and EMB Lactose Agar to score for P and check Lac. superinfecting F Lac+P- and native F Lac-P+. The rarity of singly defective segregants from a strain carrying only an F Lac+P+ episome is shown also in Table 2 for comparison. The combined transfer and segregation results indicate that there is a compatibility between native and superinfecting F Lac P such that both can exist in the same cell. However, the production of a recombinant F Lac+P+ with anomalous segregation cannot be entirely ruled out. Interaction between F Lac and F Gal. Superinfection of an F Gal strain by F Lac was studied as an example of the fate of a superinfecting F' factor carrying different genes from the native F' factor. A strain carrying the chromosomal markers Lac-Gal- (and M-Sr) and episomal F Gal+ was used as the recipient for F Lac superinfection and an F Lac+/SS strain was used as a donor. The cross was plated on EMB lactose agar containing streptomycin to eliminate the parental F Lac+. The Lac+ transfer frequency is given in Table 3 for the F' by F' cross and for an F Lac+ by Lac-F- cross. Eight Lact colonies were picked and purified by streaking on EMB lactose agar. Five of these eight Lac+ strains retained their Gal+ character after purification. The five Lac+ and Gal+ presumptive superinfected strains (designated SLac) were then tested for transfer of Gal and Lac. Gal+ transfer was determined by mating the SLac with a Gal-Fand plating on minimal galactose agar. Lac+ transfer was determined by crossing the SLac with a Lac-F- strain and plating on minimal lactose plates. The parental SLac strain was eliminated in each case by its methionine requirement. All of the five SLac strains behaved identically in their transfer characteristics; each transferred Gal+ with the very high frequency (near 100%) of F-linked markers in F' by F- crosses, but Lac+ with a very low frequency (around 0.3%). Thus the presence of F Gal appears to have either excluded the superinfecting F Lac (with the production of Lac+ chromosomal recombinants which may be transferred at low frequency) or to have suppressed transfer by F Lac. Segregation studies showed the presence of Gal- segregants but not Lac-, suggesting the incorporation of Lac+ into the chromosome in these strains, although segregation of F Lac could have been suppressed along with transfer. Of the three Lac+ Gal- strains derived from F Lac superinfection, two appeared to have replaced F Gal by F Lac, since they exhibited high frequency transfer of Lac+. F Lac may have excluded F Gal in these cases or F Gal may have been lost in a segregation event prior to F Lac addition. Superinfection experiments were also performed in which F Lac was the native F' factor and F Gal the superinfecting one. A strain with chromosomal Gal-Lac- (and MSr) and episomal F Lac+ was used as a recipient and an F Gal+/SS strain was used as the donor. The cross was TABLE 3. Transfer of Lac and Gal in F' by F' and F' by F- crosses* Donor Reiin Lac+ Gal+ eoye episome Recipient genotype transfer transfer F Lac+ F Gal+/Lac-Galh 0.3 F Lac+ Lac-Gal-F- 95 F Gal+ F Lac+/Lac-Gal - 2 F Gal+ Lac-GalFF- 99 * Crosses were carried out under standard mating conditions, and mated cultures were plated on EMB Lactose Agar in Lac+ transfer experiments and on EMB Galactose Agar in Gal+ transfer experiments. The donor Ss episomal strain was eliminated by the addition of streptomycin to the EMB agar. The F Lac+ donor was strain W3747; the F Gal+ donor W4520; the F Gal+ recipient W4520/B8; the F Lac+ recipient W3747/B8; and the F- recipient B8.

5 266 ECHOLS J. BACTERIOL. plated on EMB galactose agar containing streptomycin to eliminate the donor F Gal+. The Gal+ transfer frequency is given in Table 3 for the F' by F' cross and for an F Gal+ by Gal-F- cross. Eight Galt colonies were picked and purified by streaking on EMB galactose agar. Four of these eight Galt strains retained their Lac+ character after purification. The four Lac+ and Gal+ presumptive superinfected strains (designated SGal) were tested for transfer of Gal and Lac as described for the SLac. All four SGal transferred Galt with high frequency and Lac+ with low frequency. The stable presence of F Gal therefore is accompanied by the exclusion (with Lac+ recombination) or suppression of F Lac in the Gal± Lac+ products of F Gal superinfection, as in the case of the Gal+ Lac+ products of F Lac superinfection. In addition, a simple replacement of F Lac by F Gal may occur, since the four Gal+ Lac- products of F Gal superinfection behaved as normal F Gal strains (exhibiting high frequency transfer of Gal+). Evidence for transfer exclusion by sterile F'. In an effort to obtain some idea of the fate of the superinfecting F' factor in the case of the class of F' bacteria not fertile as recipients, the ability of a newly F-transferred phosphatase gene to direct the synthesis of the alkaline phosphatase enzyme was used as a measure of transfer. Pardee, Jacob, and Monod (1959) have shown in Hfr by F- crosses that a newly transferred Lac z (3-galactosidase) gene is almost immediately active, long before recombination and integration of the gene into the bacterial chromosome is presumed to occur. A similar activity of a phage-transferred Lac z gene has been shown, and in the case of phage-mediated transfer it has been demonstrated that Lac z genes which do not multiply appear to be active (Revel, Luria, and Rotman, 1961). Therefore, it might be expected that a superinfecting F P' which is transferred but does not multiply would still direct alkaline phosphatase synthesis. Three P+-transfer experiments were performed and alkaline phosphatase formation followed. The recipient strain in each case was Sr and P-, and, in addition, to avoid complications involving control, possessed a mutation in the R2 regulator gene leading to a loss of repression of alkaline phosphatase synthesis in the presence of inorganic phosphate (Echols et al., 1961). The donor strains were R2+, SS, and P+; as R2+ they TABLE 4. Alkaline phosphatase activity directed by newly transferred P+ gene* Donor Recipient Phosphatase activ- Stable P+ genotype genotype ity of recipient transfer after P+ transfer /O F P+ F-P Hfr P+ F-P F P+ F P/P * All crosses were carried out under standard F' by F' mating conditions; 1-ml samples were pipetted every 10 min after mating into tubes containing Merthiolate to stop further enzyme synthesis, and alkaline phosphatase assays were performed on these tubes. Streptomycin was added at 40 min to prevent further growth or phosphatase synthesis by the S' donor. The phosphatase activity figures represent the increase in alkaline phosphatase activity in the culture from 40 to 80 min. This new activity (after the addition of streptomycin) reflects entirely synthesis by the recipient strain. The "Stable P+ transfer" column gives the frequency of P+ colonies found in the recipient population by plating 100 min after mating. The F P+ donor was strain W3747; the Hfr P+ donor was K1O; the F- recipient was F15; the F P- recipient was F'26/F15. possessed a very low capacity for alkaline phosphatase synthesis relative to R2- in excess inorganic phosphate, and even this small amount of synthesis could be eliminated by the use of streptomycin. A transferred P'- gene provides a Sr R2-P+ phenotype in the recipient and should produce a high capacity for alkaline phosphatase synthesis (Ss and R2+ are not transferred with a significant frequency relative to P+ transfer by the donor strains used). The extent of phosphatase synthesis by the recipient strains after attempted P+ transfer is summarized in Table 4. The first cross of F P+ with P-F- demonstrated that a newly F-transferred P+ gene is active. The second cross of Hfr P+ with P-F- showed that newly transferred P+ genes are also active in a case in whieh many of the genes directing synthesis probably did not achieve a stable existence in the cell. (Note that stable PF clones are 60 times less frequent in the Hfr by F- cross than in the F' by F- even though the difference in new phosphatase synthesis is small.) The third cross of F P+ by F P/Pshowed a much smaller rate of alkaline phospha-

6 VO L. 85, 1963 SUPERINFECTION OF F' E. COLI 267 tase synthesis than in the F P+ by P-F- cross, paralleling the difference in stable P+-transfer frequency between the two crosses as measured by P+-colony formation. These results suggest that the class of F' bacteria sterile to F' superinfection has excluded transfer rather than prevented multiplication of transferred F'. It is also possible that a transfer of F' plus a suppression of gene activity of F-linked markers occurs, or that the superinfecting F factor is destroyed after transfer in the majority of recipient cells. DIscussIoN The experiments described above demonstrate that approximately 1% of a population of F' bacteria can acquire a superinfecting F' factor (or at least can acquire episomal genes with the transfer and segregation characteristics attributed to F-linked markers). The acquisition of the superinfecting episome may occur with the retention or the elimination of the native episome. The incompatibility of F Lac P and F Gal forms an interesting contrast to the apparent coexistence in the same cell of native and superinfecting F Lac P. There is not sufficient evidence available about the nature of F to allow very profitable speculation as to whether the incompatibility reflects metabolic competition or competition in attachment to some cellular site. Cases of interaction between F' and chromosomal and episomal F (Scaife and Gross, 1962) and between F' and a "drug resistance transfer episome" (Watanabe and Fukasawa, 1962) have been described recently. The demonstration of apparently compatible native and superinfecting episomes suggests that the low frequency of stable F-duction in superinfection experiments might reflect a low efficiency of transfer (transfer exclusion), rather than an inhibition of multiplication of the transferred superinfecting agent analogous to the immunity observed in the case of temperate phages (Bertani, 1953; Jacob and Wollman, 1953). The failure of the majority of recipient F' bacteria to show P+ gene activity for alkaline phosphatase synthesis after attempted F P+ superinfection supports this transfer exclusion hypothesis. One is then left with the problem of what determines the favored F' recipients for which transfer does take place. One possibility is that the F' cell wall is only sensitive to F-infection at a particular stage in the division cycle. ACKNOWLEDGMENTS The author wishes to thank Karen Rutherford and John Reznichek for assistance in performing these experiments, and Alan Garen and Julius Adler (as local custodian of the Lederberg collection) for bacterial strains. This work was supported by grant GM from the National Institutes of Health, U.S. Public Health Service. LITERATURE CITED ADELBERG, E. A., AND S. N. BURNS Genetic variation in the sex factor of Escherichia coli. J. Bacteriol. 79: BERTANI, G Lysogenic versus lytic cycle of phage multiplication. Cold Spring Harbor Symp. Quant. Biol. 18: CAVALLI, L. L., J. LEDERBERG, AND E. M. LEDER- BERG An infective factor controlling sex compatability in Bacterium coli. J. Gen. Microbiol. 8:72. ECHOLS, H., A. GAREN, S. GAREN, AND A. TOR- RIANI Genetic control of repression of alkaline phosphatase in E. coli. J. Mol. Biol. 3: GAREN, A Genetic control of the specificity of the bacterial enzyme, alkaline phosphatase. Symp. Soc. Gen. Microbiol. 10: HAYES, W Genetic recombination in Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 18: HIROTA, Y Mutants of the F-factor in Escherichia coli K-12. Records Genetics Soc. Am. 28:75. JACOB, F., AND E. A. ADELBERG Transfert de caracteres gen6tique par incorporation au facteur sexuel d'escherichia coli. Compt. Rend. 249: JACOB, F., AND E. L. WOLLMAN Induction of phage development in lysogenic bacteria. Cold Spring Harbor Symp. Quant. Biol. 18: JACOB, F., AND E. L. WOLLMAN Les 6pisomes, 6l6ments g6n6tiques ajout6s. Compt. Rend. 247: LEDERBERG, J Gene recombination and linked segregations in Escherichia coli. Genetics 32: LEDERBERG, J., L. L. CAVALLI, AND E. M. LEDER- BERG Sex compatibility in Escherichia coli. Genetics 37: PARDEE, A. B., F. JACOB, AND J. MONOD The genetic control and cytoplasmic expression of "inducibility" in the synthesis of,s-galactosidase by E. coli. J. Mol. Biol. 1:

7 268 ECHOLS J. BACTERIOL. REVEL, H. R., S. E. LURIA, AND B. ROTMAN iosynthesis of,3-d-galactosidase controlled by phage-carried genes. Proc. Natl. Acad. Sci. U.S. 47: SCAIFE, J., AND J. D. GROSS Inhibition of multiplication of an F-Lac factor in Hfr cells of Escherichia coli K-12. Biochem. Biophys. Res. Commun. 7: TAYLOR, A. L., AND E. A. ADELBERG Evidence for a closed linkage group in Hfr males of Escherichia coli K-12. Biochem. Biophys. Res. Commun. 5: WATANABE, T., AND T. FUTKASAWA Episomemediated transfer of drug resistance in Enterobacteriaceae. I. Interactions between resistance transfer factor and F-factor in Escherichia coli K-12. J. Bacteriol. 83: