Mol Gen Genet (1983) 192:366-372 © Springer-Verlag 1983

Site-specific Recombination Following Conjugation in Escherichia coli K-12 E.A. Birge Department of Botany and Microbiology, Arizona State University, Tempe, Arizona 85287, USA

Summary. In accord with the observations of other workers, unselected marker analysis of Escherichia coli K-12 transconjugants isolated from matings involving several different Hfr strains as donors has shown that most genetic exchanges are clustered either near the selected marker or near the origin of the transferred Hfr DNA. The present work increases the number of Hfr strains tested and shows that the clustering of the recombinational events near the origin of transfer is statistically significant. It is proposed that this clustering of genetic exchanges is due to the action of a unique recombination system (site-specific conjugal recombination or ssr) which recognizes the early transferred portion of the F plasmid and catalyzes a genetic exchange in or near the adjacent bacterial DNA. Twelve Hfr strains representing eleven different points of origin were tested, and only KL16 and Ra-1 did not demonstrate the typical clustering of genetic exchanges. Since these strains share a common transfer origin, they may represent spontaneous mutations affecting the ssr system.

mal genetic exchanges are not randomly distributed along the DNA, as would be expected with truly generalized recombination, but rather seem to occur with disproportionate frequency adjacent to the origin of D N A transfer. (Jacob and Wollman 1961; Maccacaro and Hayes 1961; Pittard and Walker 1967; Low 1968; DeHaan et al. 1972). In this communication, data are presented which confirm and extend the observations of disproportionate recombination adjacent to the origin of transfer in most, but not all, of the Hfr strains tested. Based on this genetic evidence, a model postulating a secondary, recA-independent, sitespecific recombination pathway is presented. Materials and Methods The bacterial strains used in these experiments are described in Table 1. The positions of pertinent markers and the Hfr points of origin are shown in Fig. 1. Matings were carried out for 40 rain using an H f r : F - ratio of 1:10 and then interrupted as previously described (Birge and Low 1974)

Introduction During conjugation in Escherichia coli K-12, a single strand of D N A is transferred from the Hfr donor cell into the F - recipient cell. The resulting partially diploid cell must recombine the extra D N A with the resident D N A if it is to become a true recombinant cell. Since the recipient DNA. is circular while the transferred donor D N A is linear, two exchanges must occur to produce a viable recombinant. The location of one of these exchanges is determined by the selection used and is in a position distal to the selected marker (on the side away from the transfer origin). The other exchange, however, can occur at any point proximal to the selected marker. It has been commonly assumed that both of these recombination events are catalyzed by the generalized or reeA-dependent recombination system, since recA mutant strains produce very few recombinant progeny (Low 1968; Guyer and Clark 1976) and no transcribable recombination product can be detected (Birge and Low 1974). There are conflicting reports in the literature as to the fate of Hfr markers which are transferred early. Low (1965) has reported that direct selection for the earliest Hfr markers results in the production of fewer recombinants than selection for markers transferred somewhat later. However, numerous workers have reported that the proxi-

HfrH

P4X

trp

V

KL208

~sL

/ Ral

E

D

~

PKIgl

Fig. 1. Genetic map of pertinent markers and Hfr strains used in these experiments. The tip of each arrowhead represents the earliest bacterial D N A transferred. The figure is based on the map of Bachmann and Low (1980)

367 Table 1. Bacterial strains used in these experiments. Hfr points of origin and directions of transfer are shown in Fig. I Strain

Genotype

Source or reference

EBll5

F - metE90, trpA9605(amber), thyA711, trpR55, his-85(amber), proB48, argG83, leu-62, lacI22, lacZ l18( ochre), rpsL171, azi-9, gyrA19, 2-

Derived from EB50 (Birge 1975) by two separate 5-bromouracil mutagenesis procedures

KL200

F- metB1, his-68, trp-45, pyrD34, thyAllS, thr-21, argG34, galK35, rpsLl l8, deoC2 or deo-44

K.B. low

KL195

F - like KL200 butpoIA1, arg-35 F - thi-1, thr-l, proA2, his-4, argE3, leuB6, tsx-33, ara-14, mtl-1, lacY1, galK2, glnV44, rpsL31, xyl-5, 2-

K.B. Low

F - like AB1157 but srl F- like ABl157 but recA13

A.J. Clark K.B. Low a

F- thi-1, ilv-1, argH1, metB1, rha-1, lacY1 or Z4, gal-6, rpsL8, 9, or 17, 2-, supE44 ?

A.L, Taylor via E. coli Genetic Stock Center

ABl157

JC9874 AB2463 AT753

K.B. Low

Z478

F- leuB6, proC32, purE42, K.B. Low metE70, trpE38, lysA23, rpsLl09, xyl-5, mtl-1, ara-14, lacZ36, thi-1, azi-6, tonA23, sup-45

HfrH P4X

Hfr thi-1, relA1, 2 Hfr metB1, relA l, 2-

K.B. Low"

KL208

Hfr metB1, 2-

K.B. Low

K.B. Low"

KL96

Hfr relA1, 2-

K.B. Low a

PKI9~

Hfr lae-proB, glnV44, relAl ?, colV-, colVR, 2-

JC12

Hfr metB1, purF1, lacY1, xyl-7, mtl-2, gal-6, tonA2, tsx-1, gln V44, fi-

Kahn (•968) via E. coli Genetic Stock Center K.B. Low a

Ra-I

Hfr mal-28, gln V42, fiR fl-

E. coli Genetic Stock Center"

Ra-2-1

Hfr mal-28, glnV42, fiR, 2-

Isolated for this study from Ra-1

P13(18)

Hfr his-49, cys-23, gal-5, rpsL58, fl-, Tt R, T3 R, lysogenic for phage 18

M. Schwartz

KL16

Hfr thi-1, relA1, spoT1, fl-

E. coli Genetic Stock Center"

ED1032 Hfr A(gpt-lac)5, relA1, rpsE2123, thi-1, 2-, glnV44, TP3 (transposed Ftslac ) Hfr6-1 Hfr metB1, mal-29, mtl-8, relA1, mutT2, 2R, 2

Broda et al. (1972) via E. coli Genetic Center E. coli Genetic Stock Center"

number of transconjugants increased less than 10% in 24 h), unselected genetic markers were checked by first"preparing a grid of recombinant colonies on a fresh selective medium plate, then incubating the plate for 24 h, and finally replica plating the recombinant cells onto appropriate media. The srl marker in strain JC9874 was somewhat leaky and could only be scored accurately when patches of bacteria were replicated onto MacConkey-sorbital plates which were then incubated anaerobically in a Gas Pak Jar (Baltimore Biological Laboratories). Acridine orange curing of recA transconjugants was tested by replicating grids to Y E T plates containing 25 g/ml acridine orange, growing the replicas overnight at 42 ° C, then replicating the treated cells onto the original selective medium. Roughly 67% of the Gal + patches and 90% of the Lac + patches which were still sensitive to UV radiation were also insensitive to acridine orange curing and therefore were not the result of F-prime formation. All biochemicals and agar were purchased from Sigma Chemical Company, St. Louis, Mo, Bacto Yeast Extract and Bacto Tryptone were purchased from Difco Laboratories, Detroit, M1. Results

Experiments which have suggested the existence of a secondary recombination pathway involved a minimum of two genetic markers; one located near the Hfr origin of transfer (unselected or proximal marker), and one located at some distance from the origin (selected or distal marker), as shown in Fig. 2. As noted above, it was necessary for a minimum of two genetic exchanges to occur. One exchange must have occurred at a point beyond the selected marker (region D of Fig. 2), and one must have occurred at a point on the transfer origin side of the selected marker (region P of Fig. 2). This latter exchange might have been located on either side of the unselected proximal marker (regions 1 and 2 o f Fig. 2). Therefore if the recombinant colonies were tested for inheritance of the unselected proximal marker it was possible to localize roughly the unselected genetic exchange. A series of matings of this type has been performed and the data obtained are presented in Table 2. If additional unselected markers are scored, it is possible to circumscribe more precisely the regions within which both proximal and distal genetic exchanges have occurred. While it is true that more than one exchange might occur in regions P and D, all calculations will be based on the assumption that a single exchange occurs in each region. This assumption will be validated in the D I S C U S S I O N section. Such additional analysis was performed on recombinants from the matings with HfrH, KL96, and KL208, and the data obtained are presented in Table 3. It is clear from Htr Origin

U t

~ 1

S I 2

J

" Predigree information can be found in Bachmann (1972)

(Tables 3 and 4) or by blending for two rain on a Vortex Mixer (Tables 5, 6, and 7). Recombinant cells were selected using modified minimal medium 56 (Apostolakos and Birge 1979). After two or three days incubation at 37 ° C (total

Fig. 2. Areas within which a genetic exchange may occur after conjugation. The selected marker is indicated by S, and the unselected marker is indicated by U. One exchange must occur in region D, and the other must occur somewhere within region P

368 Table 2. Unselected marker analysis after interrupted matings Hfr donor

HfrH P4X KL208 KL96 PK191 KL16 JC12

Selected distal marker

Unselected proximal marker

proB + trpA + leu + proB + leu + trpA + thyA + his + metE +

leu + leu + proB ÷ trpA + trpA + his + his + thyA + argG +

Distance from origin to proximal marker (min)

Predicted

Observed

5.2 5.2 0.~1.5 2.5 2.5 1.0 2 1 2.5

55 17 26 10 9 6 11 6 13

68 (55-79) 28 (15-45) 60 (47-72) 51 (38-65) 46 (22-72) 57 (44-70) 28 (17-41) 7 (2-16) 20 (10-33) 37a(25 51)

Percent transconjugants inheriting proximal marker

Number tested

100 60 100 99 26 100 100 100 85 99

a Selected using 50 lag/ml nalidixic acid instead of streptomycin to kill Hfr cells F - strain EBll5 was mated to various Hfr strains for 40 min. After chilling and mechanical interruption, samples were plated on selective media. The points of origin and directions of transfer of the Hfr strains are shown in Fig. 1. All matings were carried out in consecutive fashion during a two h period. Map distances are based on the map of Bachmann and Low (1980). Predicted inheritance values were conservatively calculated by using the maximum estimated distance from unselected marker to Hfr origin. If the actual distance is less than that estimated, the predicted value would be proportionately lower. The numbers in parentheses represent the 99% confidence limits assuming a bionomial distribution

Table 3. Distribution of cross-overs along the E. coli genetic map Hfr donor

% cross-overs/min occurring between Met

KL96 KL208 HfrH

Leu 1.5 6.0 "13.4

!

Pro 5.0 10.3 4.2

Lac 26.8 9.8 7.0

!

Trp 2.3 1.8 3.5

!

His 1.5 21.5 6.3

Thy 29.0

*

* 0.5

Matings were allowed to take place for 45 to 60 min, then aliquots of the culture were diluted and plated on medium selective for the indicated marker. Grids were prepared from the resulting transconjugants and replicated to test for unselected marker inheritance. The cross-over distribution was tallied separately for both proximal and distal events, and the percentage of cross-overs in each map interval was normalized by dividing by the known map distance (Bachmann and Low). The selected marker is indicated by " !", and the Hfr origin of transfer is indicated by " * "

Tables 2 and 3 that there was a tendency for clustering o f genetic exchanges near the Hfr origin o f transfer. In order to assess the statistical significance o f the clustering o f exchanges near the transfer origin, it was necessary to m a k e a c o m p a r i s o n between the observed inheritance o f p r o x i m a l m a r k e r s for the matings described in Table 2 and the percent inheritance predicted by assuming that the p r o x i m a l genetic exchange event was catalyzed by the rand o m l y acting r e c A - d e p e n d e n t enzyme system. The predicted inheritance can be obtained from the ratio of the distance between the transfer origin and the unselected p r o x i m a l m a r k e r (region 1 of Fig. 2) to the distance between the transfer origin and the selected distal m a r k e r (region P o f Fig. 2). W h e n the distance from transfer origin to unselected m a r k e r was large, it was unlikely that an origin specific event would have any substantial impact on recombination frequencies. Therefore only certain combinations o f markers have been presented in Table 2. Table 2 also demonstrates that for these combinations o f markers, the 99% confidence limits for the observed inheritance generally do not include the predicted values. As a further examination o f the significance o f the re-

sults in Table 2, the Wilcoxon test for paired cases was used to calculate the probability that the differences between the predicted and observed columns was due to chance. The resulting value, < 0.4%, indicates a highly significant difference between the paired cases. This in turn suggests that the assumption o f r a n d o m location o f proximal genetic exchanges is unwarranted. Since there did not a p p e a r to be r a n d o m l y distributed recombination events, it seemed possible that origin-specific recombination might not even require r e c A function. M u t a tions in r e c A greatly reduce, but do not entirely eliminate, recombinant p r o d u c t i o n (Guyer and Clark 1976). Therefore by plating large numbers o f cells, it was possible to obtain enough colonies for study. The results obtained are presented in Table 4. It is quite clear that the p r o x i m a l m a r k e r was inherited preferentially, even in the absence o f r e c A function. In fact, the 99% confidence limits for the r e c A values do not even include the values obtained with r e c A ÷ strains. Therefore there is a highly significant difference between proximal m a r k e r inheritance in r e c A ÷ and r e c A strains. It seemed possible that there might be some special rec o m b i n a t i o n p r o p e r t y associated with t h e s t a n d a r d Hfr

369 Table 4. Independence of proximal marker inheritance from the recA system Transconjugants from

Transconjugants/ml

% inheriting unselected marker

Number tested

Pre- Observed dicted HfrH x AB1157

2.9 x 105

24

53 leuB + (40-67)

96

HfrH x AB2463

9.4 x 101

24

79 leuB + (65-92)

65

KL96 x EB115

1.57 x 102

3

32 his + (22-42)

176

KL96 x EBll6

0.83

3

75 his + (46-99)

20

Matings were carried out in the usual manner. Cells were washed by centrifugation, and concentrated cell suspensions were spread on selective media. Transconjugants were scored as before, and recA transconjugants were also checked to be certain that they were still sensitive to UV radiation, were not susceptible to acridine orange curing (Guyer and Clark 1976), and still possessed at least one (HfrH) or two (KL96) of the original F - markers. The numbers in parentheses are the 99% confidence limits assuming a binomial distribution

Table 5. Proximal marker inheritance is not a function of the recipi-

ent strain Hfr strain

Selected distal marker

unselected proximal marker

Percent of recombinants which received proximal marker

Number of recombinants tested

Pre- Observed dicted HfrH KLI6

galK + his +

thr + thyA +

18 6

50 (3%6/) 10 (4-17)

173 169

Mating conditions were the same as those of Table 2 except that strain KL195 was used a recipient. The numbers in parentheses are the 99% confidence limits assuming a binominal distribution

transfer origins. Therefore a transposition Hfr, ED1032, was mated to KL200 and m e t B + recombinants were selected. A m o n g these, 94% were t h y A +, 86% were a r g G +, and 65% were thr + whereas only 14% t h y + would be predicted. Since ED1032 represents the forced insertion of Fts lac at an u n u s u a l site, these results indicated that any Hfr origin may be capable of preferential marker inheritance. I n point of fact, only two matings shown in Table 2 have given observed frequencies of genetic exchanges between the unselected marker and the Hfr origin of transfer for which the 99% confidence limits overlap the predicted frequency. This occurred in the cases of KL16 and HfrH, and might represent either an artifact of the mating system used or a real difference between Hfr strains. Additional observations showed that HfrH proximal markers were inherited very significantly more frequently than predicted but KL16 proximal markers were not. The results were consistent over repeated experiments and were independent of the F - strain used as a recipient (Table 5). Low (1965) has suggested that markers too close to the transfer origin do not recombine well. However if that were the problem with K L I 6, then a marker situated further from the transfer origin (srl) should be inherited more frequently than predicted. Table 6 shows that this does not occur. Moreover matings using other Hfr strains with a similarly situated proximal marker do not give similar results. KL96 and PK191 (Table 2) or P13(18) (Table 6), all of which have proximal markers as close or closer to the transfer origin than does KL16, gave preferential marker inheritance. Hfr6-1, whose origin is very precisely k n o w n (Hadley and Deonier 1979), gave 55% trp + recombinants inheriting proximal p u r E markers. Furthermore, Ra-1, which has a point of origin similar or identical to KL16, also showed a failure to give preferential marker inheritance (Table 6). Ra-1 is an unstable Hfr which is in equilibrium with a second Hfr designated Ra-2, and with an a u t o n o m o u s F strain designated R a F + (Low 1967). Guyer et al. (1980) have shown that the host strain carries two specific sex factor affinity ( s f a ) sites, a n d Low (1967) has observed that 90% of all transfer from an R a F + culture occurs from one of these sites. A test of an Ra-2-1ike Hfr isolated from Ra-1 gave results indicating unselected marker inheritance which was significantly different from the predicted value at the 95% level but not at the 99% level (Table 6). A further test of Ra-2-1 has confirmed that it displays preferential marker inheritance, while Ra-1 does not.

Table 6. Proximal marker inheritance depends upon the Hfr point of origin, not the distance from the origin to the proximal marker.

Matings were performed in the usual manner. Data for all strains except KL 16 represent the summation of two different matings Hfr strain

KL16 Ra-1 Ra-2-1 P 13(18) Hfr 6-1

Fstrain

EBI15 JC9874 EB115 JC9874 JC9874 AT753 X478

Selected marker

his + his + his + his + thr + argH + trp +

Unselected marker

thyA + srl + thyA + srl + argE + ilv + proC + purE +

Distance from origin to unselected marker (rain)

Percent of recombinants inheriting proximal marker Predicted

Observed

1.0 3.7 1.0 3.7 1.0-1.7 1.3 0.26 3.2

6 21 6 21 13 21 1.5 17

8 11 4 21 22 82 4 55

(2-18) (4-22) (0.210) (11-31) (13-31) (74-89) (0.01-7) (42-68)

Number of recombinants tested 87 89 135 118 148 200 99 99

370 There are at least two possible ways in which strains KL16 and Ra-1 might be defective for proximal marker inheritance. They might lack some sort of binding site on the F plasmid at which the proximal marker recombination is triggered (a cis-acting defect) or they might fail to produce one or more enzymes necessary for the recombination process to occur (repairable in trans). To test for positional effects, interrupted matings were performed using KL16 and HfrH as donors and EBl15 as the recipient. Selection was made either for His + or Leu + (inheritance from one Hfr strain) or His + Leu + (inheritance from two Hfr strains) recombinants. When the unselected thy marker was scored, the difference between the KL16 mating alone (8%) and the combined Hfr mating (13%) was significant at the 95% level. Therefore, it appears possible that the defect in KL16 may be due to an alteration in a diffusible product.

and that of 3 exchanges is 5.5%. Assuming an exchange frequency of 0.03, the respective probabilites are 32.2% and 1.7%. In the actual KL208 mating shown in Table 3, 37.5% of the recombinants tested had an odd number of exchanges between the unselected lac marker and trp (a 19 min interval), which would give an exchange frequency of 0.0368. Therefore the estimates of exchange frequency are reasonable. Using a frequency value of 0.037, the Poisson formula gives probabilities for one exchange in 19 min to be 34,7% and for three exchanges to be 2.8%. Thus over 90% of the exchanges have been calculated to be single ones, and the assumption of predominantly single exchanges appears to be reasonable. Similar observations of preferential marker inheritance have been made by others, but generally only one or two Hfr strains were tested in each study (Jacob and Wollman 1961 [HfrH]; Maccacaro and Hayes 1961 [HfrH]; Pittard and Walker 1967 [AB2154]; Low 1968 [HfrH, HfrC, KL25]; DeHaan etal. 1972 [HfrH, R4]). This paper increases the list of Hfr strains showing preferential inheritance with the addition of strains P4X (similar to R4), KL208, KL96, JC12, P13(18) (similar to KL25), ED1032, Hfr6-1, and PKI91. The inheritance of donor markers is even more pronounced in a recA recipient (Table 4). At first glance it might seem unusual to find any recombinants in a recA strain, but in fact Guyer and Clark (1976) have reported a twenty-fold excess of F - recombinants over the expected back mutation rate in a recA56 strain. Their estimated frequency of recombination for a recA56 strain was less than 10-6 that of a Rec + strain, when a very proximal donor marker was selected. The experiment reported in this paper is not strictly analagous, since selection was for a distal marker, providing a greater length of D N A in which unusual genetic exchanges might occur. Nevertheless, the yield of recombinants in AB2463 was reduced 3500-fold over that for ABl157 and a reduction of 190 fold over that seen in the EBl16/EB115 pair. It might be thought that the recA + locus of the Hfr donor might transfer regardless of the blending treatment. However, in the case of KL96, this would require the transfer of more than 40 min worth of D N A without the inheritance of any donor marker. This seems most improbable. It might also be suggested that the recA mutations used are both leaky. It seems unlikely that both recA1 and recA13 are equally leaky. However, if that were the case, it is difficult to imagine why that leakiness should result

Discussion

The data presented above indicate a statistically significant clustering of genetic exchanges near the Hfr origin of transfer (Table 2, Table 3) and a very low rate of exchange elsewhere. The observed frequency of genetic exchanges near the middle of the transferred D N A (Table 3) indicated that the probability of a genetic exchange occurring in this region was about 0.02 to 0.04 for each min of separation between markers, which is essentially the same as that determined by Pittard and Walker (1967). However, in the region of the selected marker or the Hfr origin of transfer, the probability was seen to increase fivefold or more. There also appears to be a slight hotspot for recombination between proB and lac which is independent of the donor or recipient used. (Table 3 and Piott and Birge, unpublished results). Before continuing, it is important to validate the explicit assmnption that predominantly single exchanges are occurring in regions P and D of Fig. 2. This can be done using the data of Table 3 and the Poisson distribution. For example, in the HfrH mating, 258 exchanges were scored among 149 recombinants. Since the distance from origin of transfer to selected marker is 31.2 rain, the probability of exchange is 0.05/min, which agrees well with the observations cited above. To test the agreement between these calculations and the observed results, the Poisson formula can be used to calculate the probability of exactly 1 or exactly 3 exchanges over an interval of 19 min (even numbers of exchanges would not be scored). Assuming an exchange frequency of 0.05, the probability of I exchange is 36.7%

Table 7. A diffusible product produced by HfrH assists recombination by KL16 DNA Donor

Selection

Transconjugants/ml recovered

Unselectedmarker phenotype scored

Observed percentage

Number tested

HfrH KL16 HfrH + KL16

leuB + his + leuB + his + leuB+his +

9 x 104 4 . 8 x 104 2.2 x l 0 s 3.6 x 104 1.2 x 104

-

8 8 (4-14) 16 ( 9 - 2 5 ) 13 ( 9 - 1 7 )

87 163 90 390

thyA + -

thyA + l e u B - l a c thyA +lac-trp thyA +

Strain EB115 was mated to the indicated donors for 30 rain. Mating mixtures were chilled and interrupted by vortexing. Mating ratios were 1 Hfr:10 F- for individual matings and 1:4 for each individual Hfr in the combined mating. Numbers in parentheses indicate 95% confidence limits

371 in a highly significant increase in proximal marker inheritance when compared to the isogenic rec + strain. Low (1965) has presented data which he interpreted as indicative of a failure of the transconjugants to recombine the earliest transferred Hfr markers. However, the conditions of his experiments Were different from those utilized here, since he selected directly for the proximal Hfr markers, rather than using them as unselected markers. Moreover, his most dramatic effect was seen with Hfr strain KL16, which has been shown to be unlike most Hfr srains in the observed pattern of unselected marker inheritance (Tables 3, 5, and 6). It is possible to construct a model which accounts for the data presented in this paper. In order to do so, however, it is necessary to postulate an unusual type of site-specific recombination. This recombination system (tentatively designated as ssr or sequence-specific conjugal recombination) must be capable of recognizing the portion of the F plasmid D N A which is transferred early by an Hfr strain and of catalyzing a genetic exchange within the homologous bacterial D N A adjacent to this F plasmid sequence or within the IS element bounding the inserted F plasmid. The former mode of action would be exactly analagous to those for the EcoB (Rosamund et al. 1979) and EcoPl (Smith 1979) enzymes, and would represent single-site-specific recombination in the terminology of Low and Porter (1978). At the present time a tack of knowledge regarding the points of origin of Hfr strains relative to the markers tested prevents a more precise localization Of the proximal genetic exchange. Preferential marker inheritance and hence the presumptive ssr system does not require intact recA function (Table 4) but is eliminated or reduced in recB strains (Low 1968; DeHaan et al. 1972; Piott and Birge unpublished observations). Since the recA protein is presumed to function early and the recB protein late during recombination (Birge and Low 1974), the ssr system can be viewed as an initiator of recombination which is not totally independent of other recombination functions. It also bears some formal resemblance to model number two of Warren and Clark (1980) which is concerned with the r e c A - i n d e p e n d e n t circularization of the ColE1 plasmid after conjugal transfer. Two Hfr strains (KLI6, Ra-a) seem to be defective in the ssr system (Table 5). It is known that the Ra-1 and Ra-2 Hfr strains resulted from integration of the F plasmid at ya sites located on the E. coli genome (Guyer et al. 1980). Since K L I 6 has essentially the same point of origin as Ra-1 (Fig. 1), it is reasonable to assume that it too is integrated at ya. This would suggest that integration at yc~might result in scr deficiency. It is known that the t n p R protein produced by yc~functions both in regulation and recombination (Reed 1981). If the protein was overproduced following transfer, it might cause hyperrecombination near the origin of transfer which would result in the unlinking of the proximal and distal markers. However the observed level of unselected marker inheritance after mating with Ra-2-1 suggests that this may not be the case. Alternatively the deficiency might be the result of the absence of an appropriate binding site for the ssr protein(s) on the portion of the F plasmid which is transferred early during conjugation, or it might be due to the production of a defective protein whose normal function is necessary for ssr activity. The fact that simultaneous matings with KL16 and HfrH leads to preferential inheritance of the

Thy + marker tends to favor the defective protein hypothesis. Berg et al. (1983) have reported that an F plasmid produces a cis-acting product which enhances Tn5 transposition, and Porter (1981) has reported enhanced recombination when F tra functions are constitutively expressed. Both of these phenomena may represent additional activities of the ssr system. In summary, evidence in favor of the existence of an ssr system is provided by the observations that F plasmid sequences are not inherited by most transconjugants (Hayes 1953), that the presence of F plasmid sequences in the recipient (which increases the extent of proximal DNA homology) does not increase recombination frequencies for selected markers (Walker and Pittard 1972), and that nearly all Hfr strains tested exhibit high recombination frequencies near the origin of transfer (Tables 2 and 5). The exceptions, strains KLI6 and Ra-1, may represent natural mutants in the site-specific process. Further investigations will be necessary to reveal whether other mutations affecting the postulated ssr recombination mechanism can be isolated. Acknowledgements. This research was supported primarily by funds from the Department of Botany and Microbiology. However, thanks are due to the Department of Bacteriology, University of Wisconsin-Madison for its hospitality during the later stages of this work. I am grateful to Barbara Bachmann, Richard Deonier, and K. Brooks Low for helpful discussions and for supplying many of the strains used in these experiments, and to George Davis and Tim McDonald for assistance in constructing strain EB115.

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C o m m u n i c a t e d by G. F i n k

Received January 13/August 18,1983