Chapter *38 



BACTERIA: RECOMBINATION 

 (II. Conjugation) 



I 



"t was found in Chapter 37 that 

 genetic recombination may occur 

 .in bacteria by means of genetic 

 transformation. The transformation process 

 has two unique features which had hitherto 

 not been encountered. The first is that the 

 donor DNA enters the recipient bacterium 

 without the intervention of any other organ- 

 ism, as was demonstrated by the infectivity 

 of naked DNA. Accordingly, although trans- 

 formation involves the genetic material of two 

 different cells it is not a typical sexual process, 

 since it does not depend upon contact between 

 donor and recipient cell. The second unique 

 feature is that the integration process leading 

 to genetic recombination occurs in the pres- 

 ence of a portion of the entire genome of the 

 donor cell and the entire genome of the 

 recipient, as a consequence of which only a 

 small segment of the penetrant donor DNA 

 replaces a small homologous segment of the 

 recipient chromosome. 



It would seem to be a reasonable hypothesis 

 that any homologous DNA may integrate, by 

 the same mechanism involved in transforma- 

 tion, provided it penetrates the cell. One can 

 therefore institute a search for other means 

 whereby DNA may be introduced into a 

 recipient cell. The present Chapter deals 

 with experiments designed to test whether or 

 not DNA passes from one bacterium to 

 another, when these are in contact. 



The first experiment ^ can be designed as 



' Based upon work of J. Lederberg and E. L. Tatum 

 (1946). 



349 



follows. A prototrophic strain (K12) of 

 E. coli is treated with a mutagen (like X rays 

 or ultraviolet light) to obtain single mutants 

 requiring different nutritional supplements in 

 order to grow. The mutagenic treatment is 

 repeated on the single, and then on the double 

 mutant auxotrophs to obtain finally two lines, 

 each different from the other by three 

 nutritional mutants, all six mutants having 

 arisen independently. One triple mutant 

 strain is auxotrophic for threonine (T"), 

 leucine (L"), and thiamin (5r) while the 

 other triple mutant is auxotrophic for biotin 

 (B-), phenylalanine (Pa-), and cystine (C-). 

 The genotypes of these two lines can be 

 given, respectively, as TLBi B+Pa^C^ and 

 T+L+B,+B~Pa C-. 



The pure fines are grown separately on 

 complete liquid medium. Then about 10^ 

 bacteria from one line are plated onto agar 

 containing complete medium, to form a lawn 

 of continuous colonies. In the case of the 

 TLBy- line, three replica plates are made 

 (see Figure 38-1), each one containing com- 

 plete medium minus a different single nutrient 

 (lacking T, L, and Bi, respectively). Oc- 

 casionally, a replica has a clone growing on 

 it, which can be shown to be due to mutation 

 to prototrophy for the nutrient missing from 

 the medium. However, such clonal growth 

 is not found in the same corresponding posi- 

 tion on all three replicas, or even on two 

 replicas, with greater than chance frequency. 

 The same results are obtained when the 

 B Pa C line is tested on appropriate repli- 

 cas. We conclude, therefore, that on rare 

 occasions single mutants to prototrophy for 

 one nutrient do occur, but that double or 

 triple mutants do not occur with detectable 

 frequency. 



Next, the same experiment is repeated, 

 except that the two triple mutant strains are 

 first mixed in the liquid medium before being 

 plated on agar containing complete medium. 

 In this case (Figure 38-2), six replicas are made 

 with medium which is complete except in the 



