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 and clustered either n
There exist many regions on the genetic map of E. coli, remarkable for very high frequency of genetic exchanges between the donor and recipient chromosome after conjugation. We call these regions fre (frequent recombination exchange). Two of them wer
The construction of plasmids which facilitate the study of interplasmidic and intraplasmidic recombination is described. In this system, a single recombination event between two mutated Ter genes on separate plasmids or on one plasmid leads to a chan
The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochel
When Escherichia coli K12(λ) lysogens are infected with heteroimmune λ phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational
It has previously been proposed, based on indirect evidence, that the Rho protein may control the expression of the rho gene. Using an in vitro system for the transcription and translation of the rho gene cloned into plasmid pBR322, we tested this hy
Escherichia coli is the model organism for which our knowledge of its regulatory network is the most extensive. Over the last few years, our project has been collecting and curating the literature concerning E. coli transcription initiation and opero
The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state o
The F′ plasmids ORF-1 (purE+tsxsproC+lac+) and F′14 (agrE+metB+ilv+) contain active regions of recombination, fre I and fre II correspondingly. The plasmid ORF-1 is stable in recF− cells. (i.e., with the RecBC pathway of recombination) and decays in
Twenty-four dnaB alleles have been ordered by deletion mapping of the Escherichia coli K-12 dnaB gene. The position of the alleles had no linear correlation with the known phenotypes of dnaB mutation: fast shut-off, slow shut-off but immediate change
Mechanism of Genetic Recombination during Bacterial Conjugation of Escherichia coli K 12 IV. Heterogeneity of Progeny of Exconjugants. Role o f D o n o r a n d R e c i p i e n t S t r a i n s S. E. Bresler, V. A. Lanzov, a n d L. R. M a n u k i a n Physico-technical Institute of the Academy of Sciences of USSR, Leningrad, USSR Received October 26, 1971
Summary. ~Iethods of clonal analysis were applied to the study of heterogeneity of the progeny after crosses of 4 donor strains (Hfr H, Hfr C, KL 16 and KL 99) with 3 recipient strains (PC 0212, AB 712 and ECK 022). Three markers were used in each cross. The distal one was the selective marker. The inheritance of two additional proximal markers characterized the heterogeneity of clones originating from particular zygotes. In most crosses the percentage of heterogeneity exceeded 30. One of the recipient strains, obtained by conjugation of the conventional strain PC 0212 with the donor I-Ifr H revealed unusual properties in respect to heterogeneity. Exconjugants derived from this recipient (ECK 022) and donor Hfr H and Hfr C had a heterogeneity index of about 5 %. I t is shown that this unusual behavior reflects a very fast process of segregation of recombinants. In crosses with the donors KL 16 and KL 99 the same recipient revealed normal indices of heterogeneity. All these data are explained assuming that there exists a specific genetic marker which determines the process of decay of merozygotes. Tentatively it is called her. Its approximate localization was deduced from specifically designed experiments, in which the heterogeneity of the progeny was found very different, when the donor KL 16 transmitted different parts of its chromosome to the recipient ECK 022. Introduction The h e t e r o g e n e i t y of p r o g e n y of single h e t e r o z y g o t e s in b a c t e r i a l c o n j u g a t i o n was first discovered b y L e d e r b e r g (1957) a n d A n d e r s o n (1958). I t was confirmed b y clonal analysis (Bresler et al., 1967 ; Wood, 1967) a n d is now a well e s t a b l i s h e d fact. Nevertheless i t r e m a i n s unclear w h e t h e r this p h e n o m e n o n is a n o b l i g a t o r y result of conjugation. Two conflicting p o i n t s of view were expressed. One (Bresler et al., 1967) affirmed t h a t h e t e r o g e n e i t y of t h e p r o g e n y is a direct consequence of t h e r e c o m b i n a t i o n mechanism. Therefore i t m u s t be quite general. The second (Wood, 1967) s u p p o s e d t h a t i t is an exclusive p r o p e r t y of t h e donor H f r H, conn e c t e d w i t h t h e fact t h a t c h r o m o s o m e t r a n s f e r in this s t r a i n has a n opposite p o l a r i t y in r e l a t i o n to t h a t p r e s u m e d for v e g e t a t i v e r e p l i c a t i o n (Curtiss, 1969). T h e aim of this s t u d y was t o d e t e r m i n e t h e origine of t h e h e t e r o g e n e i t y of r e c o m b i n a n t s . W e used four donors (two of which t r a n s f e r r e d t h e i r chromosome clockwise, t w o - - c o u n t e r - c l o c k w i s e ) , a n d t h r e e recipients. W e conclude t h a t this p h e n o m e n o n is n o t t h e privilege of some special d o n o r or recipient strain, b u t d e p e n d s on t h e c o m b i n a t i o n of t h e i r genotypes. The degree of h e t e r o g e n e i t y is a h e r e d i t a r y p r o p e r t y of t h e cell.
S . E . Breslcr et al. :
Materials and Methods Nomenclature. We used the symbols of Taylor (1970) to denote the genotypes and phenotypes of cells. For most of our markers the transcription of genotype a n d phenotype coincides. The only exception was gene pur H, the corresponding phenotype is Ade +. Bacterial Strains. Strains of E. coli K 12 used in this work are listed in Table 1. Strain Hfr H originated from the collection of Dr. F. Bonhoeffer, strains Hfr C, K L 16 and K L 99 from Dr. B. Low, strain PC 0212 from Dr. P. G. de Haan, strain AB 712 from Dr. A . L . Taylor. We are deeply grateful to these colleagues for kindly sending us these strains.
Table 1. Characteristics of bacterial strains used Strain
Classical Hfr Cavalli
K L 16
K L 99
ara t a l K lac Y leu proA thi thr str A xyl
Collection of Dr. A. L. Taylor
gal guaC his ilv lac pro A pur H thi thr trp tyr A str xyl
de H a a n (1969)
(guaC) ? his ilv (lac) ? pro A purH thi thr trp tyr A str xyl
Results of a cross Hfr H X PC 0212 with selection for Gal+Str r recombinants
The recipient strain ECK 022 was very unusual in respect to heterogeneity of its progeny after mating. I t was obtained in our laboratory b y conjugation of Hfr H with F - P C 0212 and selection of Gal+Str r recombinants. Its unusual properties are not connected with the Gal marker and its finding was a m a t t e r of chance. We can suggest t h a t the strain ECK 022 inherited from the donor Hfr H a m u t a t i o n in a DNA region controlling one of the stages of recombination, led to far reaching consequences when integrated into the recipient. Media. For cultivation of strains and conjugation we used the A P medium (aminopeptide of the Leningrad factory of medical preparations) diluted 5 fold with saline. Selection of recombinants was performed on minimal agar (Jacob and Wollman, 1961) supplemented with streptomycin and all the aminoacid needed according to well known recipes (Haan de et al., 1969). Procedure o] Conjugation. Donor and recipient cells cultivated separately on the medium AP/5 were t a k e n in the exponential phase and mixed in the ratio 1:10. The suspension was aerated for 55 minutes b y gentle shaking, diluted 1 : 50 with saline and blended to interrupt conjugation. Then the mixture was plated on selective agar in suitable dilutions. Clonal Analysis o/the Progeny o/Exeonjugants. R e c o m b i n a n t clones after primary selection were purified b y transfer of a significant p a r t of every clone on selective agar of the same composition. Then the inheritance of nonselective markers was studied. Those clones which contained at least the donor allele of one of the nonselective markers were subjected to clonal analysis. They were extracted from agar, homogenized in saline and plated on selective agar plates. Usually 10 secondary clones were used, b u t sometimes more were tested for the inheritance of nonselective markers. If after the investigation of N primary clones we found t h a t IN" out of N are mixed, t h a n the ratio N ' / N was the measure of the heterogeneity. Of course this ratio does not depend on N if the statistical error is not significant.
Recombination during Conjugation. IV
UV-lrradiation o] l~ecombinants. Each clone tested for UV-indueed segregation was resuspended in phosphate buffer (0.1 M, ph 7). UV-irradiation was performed in a petri dish by means of a baeteriocidal lamp at a distance 30 cm. Different doses were used and the measured survival of cells gave a characteristic of the dose. All procedures after UV-irradiation were performed at red light to avoid photoreactivation. Results and Discussion We i n t r o d u c e a n e x p e r i m e n t a l index to characterize the results of clonal analysis of exconjugants. The i n d e x G,n~was the percentage of m i x e d clones with at least two different p h e n o t y p e s i n relation to the n u m b e r of clones of p r i m a r y selection. I n the i n d e x G~, m denotes the n u m b e r of secondary clones, n denotes the n u m b e r of nonselective markers used. Because of the large a m o u n t of work i n v o l v e d we restricted the n u m b e r of secondary clones to 10 i n most cases a n d the n u m b e r of markers to 2. Therefore we used as a rule the i n d e x G~0. B u t for special purposes we used also indices G~0 a n d G~00. The results of e x c o n j u g a n t analysis are presented i n Table 2. The n u m b e r N of p r i m a r y clones is given for every cross to characterized the statistical error. The q u a l i t a t i v e characteristics of different p h e n o t y p e s i n the p r o g e n y of the p r i m a r y clones studied is listed i n Table 3. We used 4 donor strains a n d 3 recipients. The latter c o n t a i n e d m a n y auxotrophic m u t a t i o n s spread along the E. coli chromosome (Fig. l). F o r every cross we chose at least three markers, the distal one was the selective marker. W e always tried to make comparable crosses of the same recipient with two donors h a v i n g opposite polarities of chromosome transfer. F o r i n s t a n c e i n case of Hfr H a n d Hfr C the selective markers were respectively T r p + a n d Ade + (gene p u t H). The nonselective markers were the same i n both crosses : Thr a n d Pro A. Most of the crosses were repeated 2-3 times, a n d the value of G~ c o m p u t e d for every cross a n d averaged. I n all cases, when possible, we used different genetic markers for p r i m a r y selection (crosses 1 a n d 2, 15 a n d 16). H e t e r o g e n e i t y of the p r o g e n y was tested repeatedly i n identical crosses using Table 2. Heterogeneity of the progeny of exconjugantsa Cross No.
different selective m a r k e r s t o m a k e t h e d a t a u n a m b i g u o u s . I n crosses 1-10 we used 4 donors w i t h t h e same recipient PC 0212. I n all these cases t h e i n d e x of h e t e r o g e n e i t y Gl~0is appreciable. W e conclude therefore t h a t recipient PC 0212 is a usual strain, yielding a p r o g e n y w i t h a high degree of h e t e r o g e n e i t y . ~ o w we t u r n a t t e n t i o n t o cross 11 (I~fr C × A B 712). This case was especially interesting because in t h e case of donor H f r C, W o o d (1967) f o u n d earlier a v e r y
Recombination during Conjugation. IV
low percentage of mixed clones (in our notations he measured G~2 = 7 % in crosses with the recipients P A 309 and P A 330). We see t h a t in the cross studied b y us the heterogeneity of the p r o g e n y was similar to the value found in crosses 1-10 and the analysis revealed a diversity of genotype combinations. Thus we could n o t confirm the exclusive properties of donor strain Hfr C, but the low heterogeneity found b y W o o d could be due to some p r o p e r t y of the recipient strains. Two of our recipient strains discussed above (PC 0212 and AB 712) were practically identical in respect to heterogeneity. W e did not have at our disposal the strains used b y Wood. Therefore it was i m p o r t a n t to find a recipient strain with an unusually low capacity to form mixed clones. Such a strain called E C K 022 was obtained in our laboratory (see Materials and Methods). I t is a derivative of Hfr H and F - PC 0212. W h e n this recipient was crossed with Hfr t t and Hfr C (crosses 12-16), the index G~o was found to be very low, but in crosses with donor K L 16 and K L 99 (crosses 17-20) G~0 was quite average although lower t h a n in crosses of K L 16 with the conventional recipient PC 0212. The low index of heterogeneity can have one of two alternative explanations. 1. The merozygotes can decay v e r y fast and segregate true recombinants when still in liquid medium. 2. The merozygotes are v e r y stable and are reproduced during a long time without decay. The choice between both possibilities is easily made on experimental basis. If we find a low degree of heterogeneity it m a y be due to the first cause, i.e. to early segregation. I n this case ff we t r y a higher n u m b e r of cells for mixed genotypes, we m u s t obtain essentially the same result because t h e amounts of cells of each genotype in the mixed clone are about the same. But if we deal with a v e r y slowly decaying diploid, t h a n the main part of the cell population are merozygotes and the segregants form a minority. I n this case obviously, the more cells from the same clone are tested, the higher the probability to find a mixed clone. We performed such a test with a cross Hfr C × E C K 022, which is characterized b y a low heterogeneity index. Table 4 contains corresponding data. I n case of crosses of ttfr C with the recipient E C K 022 we find equal values of the indices G~ with different m. This is a proof of an unusually fast process of segregation in this case. F o r comparison in this table corresponding measurements with the recipient PC 0212 are presented. I n this latter case the segregation rate is normal and we see t h a t the values of indices G~ are increasing with m.
Table 4. Heterogeneity of exconjugants in the crosses between donor I~fr C and recipients ECK 022 amd PC 0212 Cross No.
6~ × ~
21 + 22 + 23 Hfr C × ECK 022 24 + 25 Hfr C × PC 0212 The duration of mating was 55 rain.
G~o G~o in %
Pro+ Thr + Pro+ Thr+
S.E. Bresler et al. :
To have an independent control that the low degree of heterogeneity of the progeny in case of the recipient ECK 022 is not due to slow segregation, we tried a method used by Curtiss (1968), based on the activation of merozygotes decay by UV-irradiation. 20 primary clones from the cross 23 with the phenotype Pro+Thr + taken at random, were submitted to UV-irradiation to a survival between 5 × 10-1 and 3 X 10-L The analysis of secondary clones (200 for every primary clone) showed that no segregation was noticed after irradiation. This experiment confirms that all primary clones of exconjugants in this case are real haploids. We can state that the recipient ECK 022 is unusual, compared with PC 0212. In crosses with the same donors Hfr H and Hfr C it segregates recombinants very fast and therefore it yields homogeneous progeny if plated after 1 hour of mating. But the same recipient, when crossed with different donors (KL 16 and K L 99), reveals the usual slow decay of the merozygotes and forms a heterogeneous progeny. We may assume that this difference in the behaviour of the merozygotes of recipient ECK 022 is connected with the ability (or inability) of donors to transmit a specific genetic locus (we shall tentatively call it "her") which determines the segregation rate of recombinants. I n experiments described earlier both donors Hfr C and Hfr H transmitted DNA fragments mainly in the region T h r - - T r p of the E. coli map (Fig. 1), but the donors K L 16 and K L 99 transmitted the region Trp--Tyr. To verify this hypothesis it was decided to transmit into the recipient ECK 022 from the donor K L 16, chromosome fragments of increasing lengths beginning with the marker Tyr till Thr. For this purpose we performed the crosses 26 and 27 (Table 5). The conjugation time was correspondingly 105 and 80 rain. Primary selection was effected using the markers Trp +, Pro +, Thr +. The results were quite clear. The heterogeneity of the clones selected for Trp + was normal as in previous rantings, but in case of primary selection for Pro + or Thr + the indices of heterogeneity were low as for crosses of the same recipient with Hfr C and Hfr H. These results are consistent with the existence of a marker het, which determines the rate of decay of merozygotes, and can be localized between the starting point of the donor Hfr C (16 min on the Taylor map) and the marker pro A (7 rain). Up to now we did not succeed in localizing this gone more precisely. All types of primary clones Pro + Thr +, Pro + Thr-, Pro-Thr + selected in the crosses 26 and 27 revealed an equally low heterogeneity. We can build a selfconsistent picture of the properties of merozygotes formed by recipients PC 0212 and ECK 022 on the basis of hot gone dosage effects. We assume that in strain PC 0212 the allele of gone hot is damaged but in the derivative strain ECK 022 it was replaced by a competent allele taken from donor Hfr H (see the origin of strain ECK 022). Then the merozygotes, resulting from crosses Table 5. Heterogeneity of exconjugants in the cross KL 16 x ECK 022 Cross
~o. 26 + 27 26 -t- 27 26+27
G~0 in %
Trp+ Pro+ Thr +
Tyr+ His+ Tyr+ His+ Trp+ Tyr+ His+ Trp+ Pro+
30 5 7
85 84 71
Recombination during Conjugation. IV
of E C K 022 with donors t r a n s m i t t i n g t h e het m a r k e r will possess a double dosage of t h e het gene. This s i t u a t i o n will result in fast segregation of r e c o m b i n a n t s a n d a low i n d e x of h e t e r o g e n e i t y . I n o t h e r cases, when t h e her m a r k e r is p r e s e n t in o n l y one c o m p e t e n t copy, its dosage is n o t excessive. Therefore the d e c a y of merezygotes a n d segregation of r e c o m b i n a n t s will proceed m u c h slowlier. This situation will m a n i f e s t a high i n d e x of heterogeneity. W h a t are t h e functions of bet ? O b v i o u s l y i t codes one of t h e enzymes i n v o l v e d in t h e d e c a y of t h e merozygote. The m a i n conclusion from this w o r k is t h a t t h e h e t e r o g e n e i t y of t h e p r o g e n y of e x c o n j u g a n t s d e p e n d s b o t h on t h e p r o p e r t i e s of t h e donor a n d recipient cells or m o r e precisely on t h e h y b r i d genetic s t r u c t u r e of t h e merozygotes. Obviously i t c a n n o t be ascribed t o some special donor s t r a i n a n d is n o t d i r e c t l y d e p e n d e n t on t h e p o l a r i t y of c h r o m o s o m e transfer.
Rderenees Anderson, T. F. : Recombination and segregation in Escherichia coli. Cold Spr. Harb. Symp. quant. Biol. 23, 47 (1958). Bonhoeffer, F. : DNA transfer and DNA synthesis during bacterial conjugation. Z. Vererbungsl. 98, 141 (1966). Bresler, S. E., Lanzov, V. A., Blinkova, A. A. : Mechanism of genetic recombination during bacterial conjugation in Eschcrichia coIi K-12. I. Heterogeneity of the progeny of conjugated cells. Genetics 56, 105 (1967). Curtiss III, R. : Ultraviolet-induced genetic recombination in a partially diploid strain of Escherichia coli. Genetics 58, 9 (1968). Curtiss III, R.: Bacterial conjugation. Ann, Rev. Microbiol. 23, 9 (1969). Haan de, P. G., Hoekstra, W. P. M., Verhocf, C., Felix, H. S.: Genetic recombination in Escherichia coli. III. Mapping by the gradient of transmission. Mutation l%es. 8, 505 (1969). Jacob, F., Wollman, E . L . : Sexuality and the genetics of bacteria. New York-London: Academic Press 1961. Lederberg, J. : Sibling recombinants in zygote pedegrees of Escherichia coli. Prec. nat. Acad. Sci. (Wash.) 43, 1060 (1957). Low, B. : Formation of merodiploids in mating with a class of Ree- recipient strains of Escherichia coli K-12. Prec. nat. Acad. Sci. (Wash.) 69, 160 (1967). Taylor, A. L. : Current linkage map of Escherichia coll. Bact. Rev. 34, 155 (1970). Wood, T. H. : Genetic recombination in Escherichia coll. Clone heterogeneity and the kinetics segregation. Science 157, 319 (1967). C o m m u n i c a t e d b y P. Starlinger S. E. Bresler V. A. Lanzov L. R. Manukian Institute of High MolecularWeight Compounds of the Academy of Sciences of USSR Leningrad 199164 USSR