Early Stages of Conjugation in Escherichia Coli ROY CURTISS, III, LUCIEN G

JOURNAL OF BACTERIOLOGY, Nov. 1969, p. 1091-1104 Vol. 100, No. 2 Copyright 0 1969 American Society for Microbiology Printed in U.S.A.

Early Stages of Conjugation in Escherichia coli ROY CURTISS, III, LUCIEN G. CARO, DAVID P. ALLISON, AND DONALD R. STALLIONS Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 Received for publication 8 September 1969

We initiated these studies to learn more about the initial events during bacterial conjugation and to optimize conditions for their occurrence. We found that cells in donor cultures grown anaerobically prior to mating have (i) a higher mean number of F pili per cell, (ii) longer F pili, (iii) a higher probability of forming specific pairs with F- cells, and (iv) a faster rate of initiation of chromosome transfer than cells grown aerobically. The growth medium for the donor culture also influences these same parameters: a rich medium is superior to a completely synthetic medium. Starvation of donor cells in buffered saline or for a required amino acid results in (i) a loss of F pili, (ii) a loss in the ability of donor-specific phages to adsorb, (iii) a loss of ability to form specific pairs with F- cells and to yield recombinants, and (iv) an increase in recipient ability. These changes occur as a function of starvation time, and at rates which are dependent on the conditions of prior growth and starvation of the donor culture. Either treatment provides a rapid method for the production of F- phenocopies from donor cultures. Resynthesis ofF pili by cells within a starved donor culture commences very soon after restoration of normal growth conditions, but full restoration of donor ability, as measured by recombinant yield, occurs at a slower rate. We found, along with other investigators, that F pili are essential for specific pair formation. We also found, however, that the presence of F pili is not sufficient for display of donor ability, nor is the absence of F pili enough for cells to exhibit recipient ability. This suggests, therefore, that one or more components, in addition to F pili, are necessary for the conversion of specific pairs to effective pairs (or for chromosome mobilization, or both) and for preventing donor cells from acting as recipients. On the basis of our results, we suggest optimal conditions for achieving high mating efficiencies.

Bacterial conjugation in Escherichia coli K-12 under the control of the fertility factor F (2-4) is initiated by the formation of specific pairs be- constitute the F-specific antigen (20), and are tween donor and recipient cells. De Haan and obligatory for specific pair formation (2, 18, 28, Gross (12) experimentally defined a specific pair 29). Brinton's group (2-4, 28, 29) and others as a donor-recipient cell union that is stable dur- (18, 21, 32) have further suggested that F pili may ing gentle dilution of the mating cultures. The function as conjugation tubes in addition to their next step is the conversion of a specific pair to an role in specific pair formation. effective pair, and it must involve the establish- Our investigations on the early stages of bac- ment of a conjugation tube or bridge between terial conjugation were prompted by a desire to donor and recipient cells that would permit sub- test these ideas, to develop mating conditions for sequent transfer of donor genetic information. achieving maximal fertility of donor cultures, Mobilization of the donor chromosome (i.e., its and to provide explanations for the loss of donor preparation for linear sequential transfer) could ability caused by starvation (11, 14, 15, 22) and occur during specific and effective pair formation by prolonged growth (6). A lengthy discussion of or after effective pair formation. These three all the published data and ideas concerning the events must precede transfer of the donor chro- early stages of bacterial conjugation has been mosome during conjugation. After the discoveries published (10) and will not be repeated here. that donor cells possess a donor-specific antigen (30) and are able to adsorb donor-specific ribo- MATERIALS AND METHODS nucleic acid (RNA) phages (24, 25) to filamen- Media. The synthetic media used were minimal tous external structures (7) called F pili (4), it was liquid (ML) and minimal agar (MA) (8, 11), minimal demonstrated that these F pili are synthesized mating medium (8, 11), and M9 (1); these were supple- 1091 1092 CURTISS ET AL. J. BACTERIOL. mented with Casamino Acids (Difco), carbon sources, mating was no more than 2 X 108/ml. All conditions and other growth factor requirements at levels previ- were kept optimum for the recovery of the maximum ously listed (11). L broth and agar (23) and Penassay possible number of recombinants. The justification for Broth and Agar (Difco) were used as complex media. these procedures and the methods for interruption of Buffered saline with gelatin (8) was used for starvation mating and recombinant selection have been pre- of bacteria. sented (11). Bacteria. The bacterial strains used to obtain the Electron microscopy and enumeration of F pili data presented in this manuscript are listed in Table 1. length and number. The general procedures for attach- The procedures for maintaining bacterial strains, for ment of the donor-specific RNA and DNA phages to isolating and characterizing mutants, and for perform- donor cells and for preparation and examination of ing controls on stability of mating-type characteristics, specimens in the Siemens-Elmiskop I electron micro- on recombinant type purity, etc., have been described scope have been described (5). F pili were specifically (11). identified by their ability to attach any of the RNA Bacteriophages and antiserum. Ultraviolet-irradiated donor phages along their lengths (4, 7) and the DNA purified T4 and T6 (2.5 X 103 ergs/mm2) were used to phage fl to their tips (5). MS-2 was the phage rou- interrupt matings. Unadsorbed phages were neutral- tinely used for this purpose, sometimes in combina- ized by phage-specific antisera obtained after repeated tion with fl. Donor phages were added to samples of injections of rabbits with purified phage as described donor cultures in the presence of 2.5 X 10-2 M KCN by Adams (1). and, after 5 to 10 min at 37 C, OS04 was added to Donor-specific RNA phages R17 (supplied by R. 0.4% final concentration. A drop of the suspension K. Fujimura), M&2 (supplied by J. Schnell), f can (sup- was then immediately placed on each of two to four plied by C. Davem), MS-2 (supplied by A. J. Clark), specimen screens for each bacterial culture being and Q,B (supplied by S. Spiegelman), and the donor- tested. After 5 to 6 min to allow bacteria to attach to specific deoxyribonucleic acid (DNA) phage fl the screen, the remaining liquid was sponged off with (supplied by June Rothman Scott) were propagated filter paper and replaced by drops of 1% phospho- on a variety of donor strains by infection of cells from tungstic acid (PTA) in 0.1 M ammonium acetate a near stationary phase culture grown in L broth. (pH 5.5). The excess PTA was removed immediately CaCl2 was added to 2.5 X 10- M at the time of and the specimen screens were then allowed to dry in infection. Other techniques and procedures were air before examination in the electron microscope. as described by Adams (1). The number of F pili per cell were determined for Mating procedures. Donor strains were grown under 50 to 200 cells per preparation by examining different a variety of conditions. The recipient parents were al- fields of the specimen screens, whereas the lengths of F ways grown with vigorous aeration (11), and matings pili were measured on projected electron micrographs. were performed in 10-ml volumes in 125-ml micro- Corrections of mean numbers and lengths of F pili fernbach flasks (Beilco) immersed, to within 5 to 10 for obscuration of F pili by cells (27) were not made, mm of the metal caps, in a waterbath at 37 C. The since there was no way to objectively calculate the donor-recipient cell ratio was between 1 :10 and 1: 20, corrections to be used. In addition, the application of and the total bacterial cell density at the beginning of such corrections would only result in relative increases TABLE 1. Bacterial strainsa

Strain Mating Relevant genotype" Derivation no. type x57 Hfr H T68 strs cycB thi O-thr ara leu proB* *met F 3000e x99 F- thr- lew- T6J str7 cyc' thi Xl2d X148 F- leu- T6r purE- trpj strr cyc' thi- x9C x313 Hfr P4X6 T68 X+ T48 thy- strs mer cyc8 O-proB proA leu ara * * lac F AB2383 x462 F- leu- proA- T6r purE- trp- lysF strr metE- cyc8 thi- X148 x493 Hfr ORll prototroph T68 T4' str8 cyc8 O-proB proA leu ara* lac F xl5c x503 Hfr OR21 prototroph T6J T4J str8 cyc8 0-proC T6 purE pyr * lac F xl5c x584 Hfr OR41 proB-lac- T68 thy- str cyc' O-thr ara leu proB... met F x354c X624 Hfr OR21 pyrA- TPr T6 T4r strt cycs x503 x679 Hfr CR34 leuc T68 str cyc' O-thr ara leu proB* met F 26 X820 F- thr- proC- T6r purE- pyr- his strt cycr x723c The nomenclature is essentially that of Demerec et al. (13), with the exceptions noted by Curtiss (9). All mutations conferring auxotrophic requirements are listed, but mutations conferring inability to utilize carbon sources have been omitted for brevity. All strains are nonlysogenic unless otherwise indicated. c See Curtiss et al. (11). Strains x493, x503, x584, x624, and X820 are all closely related, being descended from x15. d See Curtiss (8). 6 See Gross and Caro (17). VOL. 100, 1969 INITIAL EVENTS IN BACTERIAL CONJUGATION 1093 in actual mean numbers and lengths of F pili and, to a density of 108 to 2 X 108 cells/ml, the mean therefore, would not contribute to any further under- number of F standing of the role of F pili in bacterial conjugation. pili per cell increases as the conditions of growth are made more anaerobic (Fig. 2). RESULTS This has also been found for a large number of F+, F', and Hfr most of which are Relationship between growth conditions of strains, not donor listed in Table 1. The mean numbers of F pili per culture and mean number and length of F pili. An cell for 21 electron micrograph of an Hfr determinations, when 18 different cell with the RNA donor strains grown in 20 ml of L broth to 108 to phage MS-2 attached laterally to its two F pili is 2 X in shown in Fig. 1. For 108 cells/ml tubes (25 by 200 mm) were donor cells grown in L broth used, were 1.4 (range of 0.2 to 3.1) with aeration

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FIG. 1. Electron micrograph ofa portion ofan Hfr cell having two Fpili with donor-specific RNA phage (MS-2) attached laterally. Numerous type I pili are recognized by their shorter length and their inability to adsorb MS-2. Negatively stained with phosphotungstic acid. X 60,000. 1094 CURTISS ET AL. J. BACTERIOL. ix=1.5 Acids + glucose, which are much better than ML + glucose, which is better than ML + succinate, ML + glycerol, or ML + lactate, which are much better than ML + acetate. These same rankings were found for both aerated and nonaerated cultures. Of course, anaerobic growth is o 15- impossible in some of these media. The addition

LL of Casamino Acids to any synthetic medium Li 10- 0 increases the mean number of F pili per cell, but Qt 5- even with this addition and growth of the culture m to a high cell density we usually observe that less z than 50% of the cells grown in the presence of succinate, glycerol, lactate, or acetate have F pili. w 25- In measuring lengths of F pili, we noted a great I 20- variation from experiment to experiment and even 3 15- from one specimen grid to another in the same experiment. The number of F pili fragments un- _j 10- attached to cells also varied greatly. We believe Lu , 5- that these variable results were caused by the tech- wO 0 niques of specimen preparation for electron mi- LL O croscopy. However, the mean length of F pili on g 25- nlxi=5.3 C z donor cells also appeared to be influenced by the w 20- culture conditions. A generalization, based on Lu 15- observation of many preparations, is that cells - 9 L.l grown with aeration have F pili from 1 to 1.5 cell 10- lengths long and cells grown without aeration or 5- with nitrogen bubbling have F pili from 2 to 3 0 cell lengths long. 0 2 4 6 8 101 2 14 The mean numbers of F pili per cell and mean F PILI PER CELL lengths of F pili of 1.0 and 1.2 ,m, respectively, FIG. 2. Number ofF pili per cellfound after growth reported by Brinton and collaborators (2-4, 27, ofx503 (Hfr OR21) in 20 ml ofL broth in tubes (25 by 29), are lower than the values we obtained. This 200 mm) at 37 C to 108 to 2 X 108 cells/ml with aera- could be due to differences in the donor strains, tion (A), with no aeration (B), and with nitrogen bub- the cultural conditions, or the method of specimen bling (C). Values are based on an examination of 161 preparation for examination in the electron cells (A), 151 cells (B), and 132 cells (C). microscope, or to all three. Regarding this last point, we find that the addition of OS04 to the and 2.7 (range of 1.6 to 4.6) without aeration. donor cell-donor-specific phage mixture just be- The effect noted above is due to the level of dis- fore spotting it on the specimen screen results in a solved oxygen in the medium and not to a lack of "stiffening" of F pili, which results in most F pili physical agitation of cells in the nonaerated cul- protruding perpendicularly from the cell as viewed tures. The mean numbers of F pili per cell are in the electron microscope. This procedure would equally low for 20-ml aerated cultures grown in certainly minimize obscuration of F pili, in part bubbler tubes and for 10-ml cultures grown in or in toto, by the donor cell. stationary 125-ml microfernbach flasks with a Effects of F pili number on recombinant yield. fluid depth of 1 mm; furthermore, if cultures are Table 2 presents data from a representative experi- grown to densities of 5 X 108 cells/ml or higher, ment showing that donor cultures grown with the mean numbers of F pili per cell are the same nitrogen bubbling give the highest recombinant for the three culture conditions used to obtain the yields and cultures grown with aeration the lowest data presented in Fig. 2. yields. These results can be accounted for by the The mean number of F pili per donor cell is also increased number or length of F pili, or both, on influenced by the growth medium. With regard to cells grown under the more anaerobic environ- F piliation, L broth, Penassay Broth, ML + 0.5 % ments or by the influence of some unknown Casamino Acids + glucose, or M9 + 0.5 % factor. Casamino Acids + glucose are equivalent to and Relationship between mean F pili number and better than ML + 20 naturally occurring amino specific pair formation. The involvement of F pili acids + glucose (11) or ML + 0.1% Casamino in specific pair formation is clearly seen by ob- VOL. 100, 1969 INITIAL EVENTS IN BACTERIAL CONJUGATION 1095

servation of early stages of conjugation in the TABLE 2. Effects on recombinant yield of different electron microscope (2). Figure 3a is an electron growth conditions for the Hfr parenta micrograph of a presumed specific pair. The Hfr Recombinants per donor cell is connected to the F- cell by two F pili. In cell (X 100) Mean no. Fig. 3b, an Hfr cell has made contact with two Growth condition of___F__pili for Hfr parent per______o pillb different F- cells. Examination of suspensions of proC+ purE+ Pyr+ pecli donor cells grown in a rich medium without aera- strr strr strr tion soon after and recipient cells mixing reveals With aeration... 2.4 23 2.7 0.7 that donor cells frequently (about 50%) are con- Without aera- nected to more than one F- cell by F pili. Also, tion ...... 7.8 52 8.8 1.3 more than one F pilus is usually observed to span With nitrogen. . 11.6 69 7.9 1.5 the distance between Hfr and F- cells. The data presented in Fig. 4 show that the final Strain x503 (0-proC purE pyr ... ) grown in L frequency of specific pairs formed, as indicated broth under the conditions indicated was mated by recombinant yield, is related to the mean num- with x820, which had been grown with aeration in ber of F pili per cell for each culture. This relation- L broth. At 5 min after commencement of mating, ship was true in a the mating mixtures were diluted 1:100 into pre- variety of experiments of this warmed L broth to prevent further specific pair type, in which various donor strains, media, and formation. The matings were interrupted after 45 growth conditions were used. [It should be noted min by dilution into ultraviolet-irradiated T6, that, since the probability of integrating a trans- followed immediately by agitation on a vortex ferred donor marker is about 0.5 (19), the actual mixer. The frequency of inheritance of the proxi- number of specific pairs formed is about twice the mally transferred Hfr proC+ marker is reduced number of recombinants inheriting a proximally because this marker is about 0.1 min of transfer transferred Hfr marker.] Donor cultures having time from the origin of chromosome transfer for cells with low mean numbers of F per Hfr OR21 (16, 26, 31). pili cell b usually gave continuous gradual rise in the Determined by electron microscopy. frequency of specific pairs after the initial rapid rise in the frequency of specific pairs. This was that donor cultures grown without aeration give undoubtedly due to increase in the Hfr cell titer earlier first-entry times for both proximally during the mating (17) or to the continual syn- and more distally transferred donor markers thesis of F pili during the experiment by donor than do donor cultures grown with aeration. cells lacking F pili at the inception of mating (27), It seems, therefore, that donor cells grown without or to both. Analysis of the data from all these aeration are capable of converting specific pairs experiments did not reveal any discernible differ- to effective pairs or of bringing about chromo- ences in the initial rates of specific pair formation some mobilization more rapidly than donor that were related to mean numbers of F pili per cells grown with aeration, or both. cell, growth conditions, etc. (The rate of specific Effects of starvation on ability of donor strains pair formation is defined as the percentage in- to yield recombinants and to attach donor-specific crease in number of specific pairs per unit time in RNA phages. Fisher (14) has shown that donor relation to the maximum number of specific pairs strains lose their ability to yield recombinants attained.) We believe, therefore, along with other when starved for extended periods in buffered investigators (2, 18, 28, 29), that the ability to saline. The data presented in Fig. 6 indicate form specific pairs is solely dependent on the pres- that Hfr cells grown with aeration lose donor ence of F pili and, furthermore, that it is little ability, as a function of starvation time, more affected by differences in F pili length and other rapidly than do Hfr cells grown without aeration, cellular structures or by capabilities resulting and that aeration during starvation results in a from the various cultural conditions employed. more rapid loss of donor ability than does no Relationship between growth conditions of donor aeration. The data in Fig. 7 reveal that loss of culture and time of initiation of chromosome trans- donor ability during starvation in buffered saline fer. Interrupted matings with donor cultures probably required metabolic activity of the Hfr grown under different conditions were conducted cells since the rate of loss is reduced at 2 C or to determine whether the differences in donor with nitrogen bubbling. The data in Fig. 6 and fertility (Table 2) could be related to something 7 also confirm our previously reported observa- other than specific pair-forming ability. A variety tion that donor ability is not immediately re- of Hfr donors were employed in these studies, and stored upon return of starved Hfr cells to media the results from one experiment are presented in that permit growth (11), since the mating media Fig. 5. It appears from this and other experiments used did allow growth of the Hfr parent. 1096 CURTISS E AL. J. BACTERIOL.

FIG. 3a. Electron micrograph ofpresumed specific pair between an Hfr cell (bottom) and an F- cell. As in Fig. 1, MS-2 was used to "stain" Fpili. The specimen was prepared soon after mixing the Hfr and F- cells. Two Fpili have been used to make contact with a single F:;- cell. X 28,000.

Figure 8 presents data showing that, as a strains is very effective in causing loss of F pili; function of starvation time in buffered saline, uracil starvation is less effective; and thymine Hfr cells lose the ability to attach the RNA starvation has no effect. (ii) For amino acid- phage R17. Since F pili contain the attachment requiring donor strains, the rate of F pili loss is sites for RNA phages (4, 7), we examined the dependent on both the growth and starvation effects of starvation of donor cultures on F pili. media. [Conditions for most rapid to least rapid Loss ofF pili during starvation of donor cultures. rates of F pili loss are: growth in ML (or M9) + Tables 3 and 4 present representative data on 0.5% Casamino Acids + 0.5% glucose with loss of F pili for different starvation conditions starvation in ML + 0.5% glucose is better than of Hfr cultures. Additional data on F pili loss, or growth in ML + 0.5 % Casamino Acids + 0.5 % the genetic consequences of F pili loss, for other glucose with starvation in buffered saline, which starvation conditions are presented in Fig. 6, 7, is better than growth in ML + 20 amino acids + and 9 and by Curtiss et al. (11). Results from 0.5% glucose with starvation in ML + 19 amino numerous studies on F pili loss during starvation acids (the essential amino acid being omitted) + lead us to the following conclusions. (i) Amino 0.5 % glucose, which is slightly better than growth acid starvation of amino acid-requiring donor in L broth or Penassay Broth with starvation in VOL. 100, 1969 INITIAL EVENTS IN BACTERIAL CONJUGATION 1097

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FIG. 3b. Single Hfr cell (right) after contact with two F- cells, each connected by two F pili. X 25,600. buffered saline.] (iii) Cultures grown with aera- The observations reported in the previous two tion lose F pili at a fast rate, whereas culture sections lead to the predictions that, in addition grown without aeration show a lag before a fast to loss of F pili, starvation of donor cultures rate of F pili loss is achieved. (iv) Aeration should cause a loss of specific pair-forming ability during starvation results in the most rapid rate and might cause an increase in recipient ability of F pili loss; cultures starved without aeration [formation of recipient phenocopies (6)]. This lose F pili more slowly, but at a more rapid rate last prediction also follows from Brinton's (2) than nitrogen-bubbled cultures. (v) The rate of observation that recipient phenocopies prepared F pili loss is greater at 37 than at 2 C. (vi) There by aerated, overnight growth of donor strains is no relative increase in the number of free F in broth lacked F pili. The data presented in pili in the suspending medium during starvation. Fig. 9 demonstrate that these predictions are These observations indicate that the loss of F correct. Similar results were obtained in other pili is probably dependent on metabolic ac- experiments of this type. tivities of the starving donor cells rather than Note that loss of donor ability can occur at a on physical forces. more rapid rate than loss of F pili (Fig. 9A), Changes in donor ability and recipient ability although this is not always the case (Fig. 9B). as a function of starvation time of the Hfr culture. Since there is a strong correlation between the 1098 CURTISS ET AL. J. BACTERIOL.

conditions leading to most rapid loss of F pili 100- lead also to the most rapid loss of donor ability and to the most rapid increase in recipient ability. t14J Thus, the most dramatic effects are observed for amino strains grown 80- 0 0 acid-requiring Hfr in ML 0 a. (or M9) + 0.5 % Casamino Acids + 0.5% z glucose with aeration at 37 C and starved in ML 0 0 -J lo (or M9) + 0.5% glucose with aeration at 37 C w c 60- (Fig. 9B). z0 0 Resynthesis of F pili and restoration of donor 0 * 0 a 0 ability upon termination of starvation conditions. z Figure 10 presents data from an experiment that w showed the resynthesis of F pili as a function o 40- w a. of time after restoration of growth conditions by addition of 0.5% Casamino Acids to the .4 starved culture. Short F pili began to appear r- after 5 min and the "normal" number of F pili

Mean numbers ofF pilh per cell were 1.0for the aerated 0. and 2.2 for the nonaerated xS03 culture. 4 / , presence of F pili and specific pair-forming ability, it must be concluded that, in addition to causing a loss of F pili and specific pair-forming ability, starvation can reduce donor ability by some other 2 / + means. The other reason(s) for loss of donor ability during starvation could be the loss of some function or potential necessary for conversion of specific pairs to effective pairs or for 0 initiating and continuing chromosome transfer, 0 2 4 6 8 10 12 14 16Y -18r or for both. TIME OF INTERRUPTION (min) In all of our experiments, recipient ability of FIG. 5. Kinetics of recombinant formation in mat- Hfr cultures did not begin to increase until after ings with Hfr OR11 grown with (solid lines and solid 1 to 2 hr of starvation. Thereafter, further starva- circles) and without (dashed lines and open circles) tion resulted in a rapid increase in recipient aeration in L broth. The same X462 (F- proA- lew- T6- ability. Eventually, in all experiments, the re- strr) culture grown in L broth with aeration was em- cipient ability of the starved Hfr cells equaled ployed for the two interrupted matings performed the recipient ability of an F- derivative of that simultaneously. The mean numbers of F pili per x493 same Hfr (Fig. 9B). It is evident from these (Hfr OR11) cell were 2.4 and 1.1 for the nonaerated results that the absence of F is not and aerated cultures, respectively. The times of first pili probably appearance of donor markers in recombinants were solely responsible for ability of "donor" cells to 1.0 min for proA+ and 7.9 min for leu+ in the mating act as recipients. with the nonaerated x493 parent and 3.4 min for proA+ We have found, in experiments of the type and 10.6 min for leu in the mating with the aerated presented in Fig. 9, that the growth and starvation x493 culture. VOL. 100, 1969 INITIAL EVENTS IN BACTERIAL CONJUGATION 1099 loo - thus reconciling the difference in rate of reap- *\*AA pearance of F pili under the two conditions. As indicated by the data in Table 4, the resynthesis of F pili by Hfr cells in amino acid-starved \\\B cultures depends only on the ability of cells to * 0 synthesize protein and not on the ability to synthesize DNA. >_ \ \Figure 11 presents data from an experiment w \ *\ designed to determine whether resynthesis of F pili and return of specific pair-forming ability \ U_ \following restoration of growth conditions for a 1 \ starved Hfr culture are paralleled by a concom- z lo0- ..itant return of full donor ability as evidenced 0 \ \ by the formation of recombinants. The formation of recombinants requires that the donor be capa- > \ \ ble of specific pair formation, effective pair formation, chromosome mobilization, and chromosome transfer. In comparing the data in c Fig. 10 and 11, it is apparent that restoration of * full donor ability takes a longer period of time after restoration of growth conditions for the

D . 100 0-__ 1L, \ L - - - - - o L~~~~~~~~~~, 0 2 3 4 5 A TIME OF STARVATION (hr) AT 37T FIG. 6. Loss ofdonor ability as afunction ofgrowth condition, starvation condition, and time. x57 (Hfr thr+ leu+ thi- strr) was grown in Penassay Broth to about 108 cells/ml, either with or without aeration and was suspended in buffered saline for starvation either with >z\ or without aeration. At the times indicated, samples of D the Hfr cultures were mixed with x99 (F- thr leir tht- u strr) in minimal mating medium containing threonine, \ leucine, thiamine, aspartic acid, and glucose. After 40 \ min, the matings were interrupted by dilution and agita- m 10 tion on a vortex mixerfor 60 sec. Thefrequencies ofthr+ \ leu+ strr recombinants were 1.5 and 2.4% in the matings u with unstarved aerated and unstarved nonaerated Hfr \ parents, respectively. (A) x57 grown and starved with- > out aeration. (B) x57 grown without aeration and ' starved with aeration. (C) x57 grown with aeration and a:\ starved without aeration. (D) x57 grown and starved with aeration. of growth conditions. However, "normal" F c pili lengths were not achieved until 30 to 40 min after starvation was ended. These results contrast with those obtained by Brinton (2) 0 2 3 4 5 6 and Novotny et al. (27), who demonstrated that TIME OF STARVATION (hr) AT 37°C maximum numbers of F pili reappeared within FIG. 7. Loss ofdonor ability as a function ofstarva- 4 to 5 min after their removal by blending. This tion condition and time. xS7 (Hfr H) was grown with is not too surprising: it has been shown that aeration in Penassay Broth and was starved in buffered does not inhibit of saline by nitrogen bubbling at 37 C (A), by aeration chloramphenicol resynthesis 2 C and by aeration at 37 C (C). Other methodsat F Fpilipiliafterafter shearing,shearing, which suggests thatthat F pilipili were(B),as describedfor Fig. 6. Thefrequency of thr+ leu+ are assembled from presynthesized subunits strr recombinants in the mating with the unstarved Hfr (2). In the case of amino acid-starved cultures, H culture immediately after suspension in buffered such subunits would first have to be synthesized, saline was 3.1%. 1100 CURTISS ET AL. J. BACTERIOL. recombinants than of thr+ recombinants for matings of 33-min duration. The data in Fig. 11 reveal that the growth time needed to restore normal levels of purE+ recombinants is longer than that required for thr+ recombinants, so donor cells that have resynthesized F pili and are thus capable of specific pair formation are apparently still not all capable of immediately initiating chromosome transfer. These donor cells are therefore blocked either in the con- w version of specific pairs to effective pairs or in

0 some later step involving mobilization of the Hfr chromosome for transfer. z 10 DISCUSSION a: One of the principal aims of this study was to z develop optimal methods for achieving maximal w recombinant yields during conjugation in E. coli K-12. Our data indicate that donor cells grown anaerobically have (i) a higher mean number of F pili, (ii) longer F pili, (iii) a higher probability of forming specific pairs with F- cells, and (iv) the ability to initiate chromosome transfer more rapidly than cells grown aerobically. The growth

TABLE 3. Loss of F pili as a function of starvation conditions and time

O 2. 3. 4. I1,i1 5 6 No. of F pili per cell after TIME OF STARVATION (hr) AT 37°C hours of treatment Medium .- FIG. 8. Increase in percentage of unadsorbed phage R17 as a function of time of starvation of Hfr H. x57 0 1 2 3 was grown in L broth with aeration to S X 108 cells/ml and then suspended in buffered saline and starved with Expt la aeration. After the indicated times ofstarvation, 0.1-ml M9 + Casamino Acids samples were added to 0.3 ml of RI7 (at a final multi- + glucose ...... 2.6 2.9 plicity of 1.0) in L broth containing 2.5 X 1J0 M Buffered saline . 2.6 1.8 0.83 0.35 CaCl2 and were incubated at 37 Cfor 15 min. The bac- M9 + leucine + glucose 2.6 0.43 teria and adsorbed phage were sedimented with a Beck- M9 + glucose ...... 2.6.. . 0.025 man microfuge, and the supernatant fluids were assayed for unadsorbed R17. Expt. 2b ML + arginine + ura- Hfr than is needed for resynthesis of F pili. cil + glucose...... 3.3 2.2 Recombinant frequencies similar to those ob- ML + arginine + glucose...... 3.3 1.4 tained in matings with unstarved Hfr cultures ML + uracil + glucose 3.3 0.13 were 40 to 50 of conditions not achieved until min ML + glucose...... 3.3 0.085 that would permit growth had elapsed prior to commencement of mating. The time needed for Strain x679 (Hfr CR34 leu-) was grown with restoration of normal donor ability is probably gentle aeration in M9 + 0.5% Casamino Acids + longer than this, in that some recovery could 0.5% glucose to about 2 X 108 cells/ml, and was have occurred during the 33-min mating period then sedimented by centrifugation, washed gently, in those donor cells capable of specific pair and suspended in the media indicated. The cells formation during the first 5 min of mating. In were gently aerated in these media, and samples with the unstarved Hfr, the Hfr thr+ were withdrawn periodically for enumeration of matings F pili. and purE+ markers appear first in recombinants bStrain X624 [Hfr OR21 pyrA- (requires argi- after 8 and 23 min of mating, respectively. There- nine and uracil)] was grown in ML + 0.5% Casa- fore, if there is any delay in the initiation of mino Acids + uracil + 0.5% glucose. The culture chromosome transfer after specific pairs have was aerated for all but the last 45 min of growth. formed, there will be a greater loss of purE+ Other methods were as for experiment 1. 4) > i o o 5 - 00 PsUi Ui

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1.L. -,/ A, .1 , , \, \, B 0 1 2 3 4 5 6 0 1 2 3 4 5 6 TIME OF STARVATION (tw) AT 3rC FIG. 9. Losses ofFpili and donor ability and increase in recipient ability as a function ofstarvation time ofHfr cultures. (A) Hfr CR34 (x679) was grown in L broth with mild aeration at 37 C and was starved in buffered saline with aeration. (B) Hfr OR21' (X624) was grown in ML + 0.5% Casamino Acids + uracil + 0.5% glucose with mild aeration at 37 C and was starved in ML + 0.5% glucose with aeration. Donor ability was determined by selecting purE+ strr (A) or purE+ cycr (B) recombinants in matings of50-min duration with x148 in L broth (A) or with x820 in ML + Casamino Acids + adenine + uracil + glucose (B). The mating mixtures were diluted 1:100 5 min after matings commenced to prevent further specific pair formationi. Matings were interrupted with ultraviolet-irradiated T6. Recipient ability was determined by selecting leu+ met+ thy+ (A) orpyrA+ str, (B) recombinants in 40-min matings between an excess of the starved Hfr culture and Hfr P4X6 (x313) (A) or Hfr ORII (x493) (B). The matings were interrupted by vortex mixing (A) or ultraviolet-irradiated T4 (B). In part A, the lowest recombination frequency was arbitrarily used as a relative recipient ability of1.0. The Hfr P4X6 cultures for the 5- and 6-hr matings were inadvertently lost. In part B, an F- derivative (x943) ofX624 was mated with Hfr ORIJ (x493) at 0, 4, and 6 hr with selection ofpyrA+ strr recombinants, and the mean recombinant frequency in these matings was used to indicate a relative recipient ability of100. medium for the Hfr culture also influences these titer at the inception of mating. Such recom- same parameters, with a rich medium being binant yields imply that almost every Hfr cell superior to a completely synthetic medium. in the mating mixture has transferred chromo- We have adopted a protocol, based on these somal material to one or more F- recipients. results, for growth of donor cultures to achieve Starvation of donor cells in buffered saline or maximal fertility during bacterial conjugation. for a required amino acid results in (i) a loss of Donor strains are grown for eight or more F pili, (ii) a loss in the ability of donor-specific generations in either L broth or ML (or M9) + phages to adsorb, (iii) a loss of ability to form 0.5% Casamino Acids + 0.5% glucose, as 20-ml specific pairs with F- cells and to yield recom- nonaerated cultures in tubes (25 by 200 mm) binants, and (iv) an increase in recipient ability. at 37 C, to cell densities of 2 X 108/ml or higher. These changes occur as a function of starvation Appropriate samples of such donor cultures are time, and the rates at which they occur are added to a final density of 107 to 2 x 107/nml to the dependent on the conditions for growth and F- culture in a total volume of 10 ml contained in starvation of the donor culture. We believe that a 125-ml microfernbach flask at 37 C. As previ- these changes require some metabolic activity, ously shown (11), the F- parent should be grown since they occur most rapidly when the culture for eight or more generations with vigorous aera- is starved for a required amino acid with aeration tion to achieve maximal fertility. Use of these pro- at 37 C rather than when the culture is starved cedures with isogenic parents results in recom- for all metabolites, is starved under anaerobic binant frequencies for proximally transferred conditions, or is starved at 2 C. Hfr markers of 50 to 150%, based on the Hfr Resynthesis of F pili commences very soon VOL. 100, 1969 INITIAL EVENTS IN BACTERIAL CONJUGATION 1103

after restoration of growth conditions to a starved Hfr culture (about 5 min). Normal numbers and lengths of F pili are found after - about 20 and 35 min of growth, respectively. Full restoration of donor ability does not occur as rapidly, however, which suggests that starva- 0 21.0- tion has affected some other function necessary for initiation of chromosome transfer. In this Q8- respect, it should also be noted that donor ability

IL Q6- as measured by recombinant production is usually lost more rapidly during starvation than are F On the other hand, loss of F pili does 4i pili. 0.2:0.- not immediately result in increased recipient 0i ability. All of these results are most easily inter- preted if we assume that one or more components, in addition to F pili, are needed to convert specific pairs to effective pairs (for chromosome mobilization, or both) and to prevent donor cells from acting as recipients. Thus, the presence 0.1 of F pili would not be sufficient to guarantee 0 5 1b 15 20 25 30 donor ability, nor would their absence be enough TIME AFTER ADDING CASAMINO ACIDS (min) for a cell to display recipient ability. FIG. 10. Resynthesis of F pili after restoration of growth coniditions to a x679 (Hfr CR34 leu-) culture ACKNOWLEDGMENT that had been starvedfor 2 hr in M9 + glucose medium. This investigation was sponsored by the U.S. Atomic Energy X679 had been grown in M9 + 0.5% Casamino Acids + Commission under contract with the Union Carbide Corporation. 0.5% glucose prior to the 2-hr starvation period. LITERATURE CITED 1. Adams, M. H. 1959. Bacteriophages. Interscience Publishers, Inc., New York. 100 2. Brinton, C. C., Jr. 1965. The structure, function, synthesis CASAMiINO 80 ACIDS and genetic control of bacterial pili and a molecular model DE for DNA and RNA transport in gram-negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 3. Brinton, C. C., Jr. 1967. Contributions of pili to the specificity of the bacterial surface, and a unitary hypothesis ofconjugal E 40 infectious heredity, p. 37-70. In B. D. Davis and H. J. z

z Vogel (ed.), The specificity of cell surfaces. Prentice-Hall, Inc., Englewood Cliffs, N.J. 4. Brinton, C. C., Jr., P. Gemski, Jr., and J. Camahan. 1964. A 20 new type of bacterial pilus genetically controlled by the fertility factor of E. colU K-12 and its role in chromosome transfer. Proc. Nat. Acad. Sci. U.S.A. 52:776-783. 5. Caro, L. G., and M. Schnos. 1966. The attachment of the 10~~~~ male-specific bacteriophage fl to sensitive stains of Esch- erichia coli. Proc. Nat. Acad. Sci. U.S.A. 56:126-132. 6. Cavalli, L. L., J. Lederberg, and E. M. Lederberg. 1953. An

6 --,- infective factor controlling sex compatability in Bacterium 0 '180 190 200 210O 220 230 240 col. J. Gen. Microbiol. 8:89-103. TIME MATINGS BEGAN (min) 7. Crawford, E. M., and R. F. Gesteland. 1964. The adsorption FIG. 11. Loss and restoration ofdonor ability. x584 of bacteriophage R-17. Virology 22:165-167. 8. Curtiss, R., III. 1965. Chromosomal aberrations associated was grown in ML + 0.5% (Hfr OR41 proTB thy-) with mutations to bacteriophage resistance in Escherichia Casamino Acids + thymine + 0.5% glucose with coli. J. Bacteriol. 89:28-40. gentle aeration at 37 C. The cells were starved in ML + 9. Curtiss, R., III. 1968. Ultraviolet-induced genetic recombina- thymine + glucose with aeration for 180 min, at which tion in a partially diploid strain of Escherichia coll. Gen- time Casamino Acids were added to 0.5% final concen- etics 58:9-54. tration. X820 (F- thr- purE- T6r strr) was used as the 10. Curtiss, R., III. 1969. Bacterial conjugation. Annu. Rev. recipient parent in matings that were interrupted after Microbiol. 23:69-136. 33 min by ultraviolet-irradiated T6. The mating mix- 11. Curtiss, R., HI. L. J. Charamella, D. R. Stallions, and J. A. Mays. 1968. Parental functions during conjugation in tures were diluted 1:100 after 5 min to prevent further Escherichia coli K-12. Bacteriol. Rev. 32:320-348. specific pair formation. The control mating (prior to 12. De Haan, P. G., and J. D. Gross. 1962. Transfer delay and starvation ofthe Hfr parent) yielded 61% thr+ strr and chromosomal withdrawal doring conjugation in E. coil. 27% purE+ strr recombinants. Genet. Res. 3:251-272. 1104 CURTISS ET AL. J. BACTERIOL.

13. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 24. Loeb, T. 1960. Isolation of a bacteriophage specific for the 1966. A proposal for a uniform nomenclature in bacterial F+ and Hfr mating types of Escherichia colt K-12. Science genetics. Genetics 54:61-76. 131:932-933. 14. Fisher, K. W. 1957. The nature of the endergonic processes in 25. Loeb, T., and N. D. Zinder. 1961. A bacteriophage containing conjugation in Escherichia coli K-12. J. Gen. Microbiol. 16: RNA. Proc. Nat. Acad. Sci. U.S.A. 47:282-289. 136-145. 26. Low, B. 1966. Low recombination frequency for markers very 15. Fisher, K. W. 1966. Amino acid deprivation and its effect on near the origin in conjugation in E. colt. Genet. Res. 6:469- mating ability in Escherichia coll K-12. Genet. Res. 8:115- 473. 118. 27. Novotny, C., J. Carnahan, and C. C. Brinton, Jr. 1969. 16. Glansdorff, N. 1966. Pseudoinversions in the chromosome of Mechanical removal of F pili, type I pili, and flagella from E. coli. Genetics 55:49-61. Hfr and RTF donor cells and the kinetics of their reap- 17. Gross, J. D., and L. G. Caro. 1966. DNA transfer in bacterial pearance. J. Bacteriol. 98:1294-1306. conjugation. J. Mol. Biol. 16:269-289. 28. Novotny, C., W. S. Knight, and C. C. Brinton, Jr. 1968. 18. Ippen, K. A., and R. C. Valentine. 1967. The sex hair of E. Inhibition of bacterial conjugation by ribonucleic acid and colt as sensory fiber, conjugation tube, or mating arm? deoxyribonucleic acid male-specific bacteriophages. J. Biochem. Biophys. Res. Commun. 27:67-680. Bacteriol. 95:314-326. 19. Jacob, F., and E. L. Wollman. 1961. Sexuality and the genetics 29. Novotny, C., E. Raizen, W. S. Knight, and C. C. Brinton, Jr. of bacteria. Academic Press Inc., New York. 1969. Functions of F pili in mating-pair formation and 20. Ishibashi, M. 1967. F pilus as f+ antigen. J. Bacteriol. 93:379- male bacteriophage infection studied by blending spectra 389. and reappearance kinetics. J. Bacteriol. 98:1307-1319. 21. Knolle, P. 1967. Evidence for the identity ofthe mating-specific 30. Orskov, I., and F. Orskov. 1960. An antigen termed f+ occur- site of male cells of Escherichia coli with the receptor site of ring in F+ E. coli strains. Acta Pathol. Microbiol. Scand. an RNA phage. Biochem. Biophys. Res. Commun. 27:81- 48:37-46. 87. 31. Pittard, J., and E. M. Walker. 1967. Conjugation in Escherichia 22. Krisch, R. E., and M. J. Kvetkas. 1966. Inhibition of bacterial coli: recombination events in terminal regions of transferred mating by amino acid deprivation. Biochem. Biophys. Res. deoxyribonucleic acid. J. Bacteriol. 94:1656-1663. Commun. 22:707-711. 32. Valentine, R. C., P. M. Silverman, K. A. Ippen, and H. Mor- 23. Lennox, E. S. 1955. Transduction of linked genetic characters bach. 1969. The F pilus of Escherichia colU. Advan. Micro- of the host by bacteriophage P1. Virology 1:190-206. biol. Physiol. 3:2-52.