Stanford Medical Bulletin 



methodology of microbial genetics. Once hav- 

 ing connected adaptive variation with gene 

 mutation (78), we could proceed to exploit 

 these systems for the detection ol specific geno 

 types in very large test populations. The geno- 

 types of interesl may arise, as in the previous 

 examples, by imitation: the most extensive 

 studies of the physiology ol mutation now use 

 these methods tor precise assay. For, in order 

 to count the number oi mutants of a given 

 kind, it suffices to place large numbers of bac- 

 teria into selective media and count the surviv- 

 ing colonies which appear after incubation. In 

 this way, mutation rates as low as one per io H 

 divisions can be treated in routine fashion. 



GENETIC RECOMBINATION IN BACTERIA 



The selective isolation of designed genotypes 

 is also the most efficient way to detect genetic 

 recombination. For example, the sexual mecha- 

 nism of Escherichia coli was first exposed when 

 prototrophic (nutritionally self-sufficient) re- 

 combinants arose in mixed cultures of two 

 auxotrophic (nutritionally dependent) mu- 

 tants (35, 57, 84). At first only one recombin- 

 ant appeared per million parental bacteria 

 and the selective procedure was quite obliga- 

 tory. Later, more fertile strains were discov- 

 ered which have been most helpful to further 

 analysis (45, 51). This has shown that typical 

 multinucleate vegetative bacteria unite by a 

 conjugation bridge through which part or all 

 of a male genome migrates into the female cell 

 (43). The gametic cells then separate. The ex- 

 conjugant male forms an unaltered clone, sur- 

 viving by virtue of its remaining nuclei. The 

 exconjugant female generates a mixed clone 

 including recombinants (46, 1). Wollman, 

 Jacob, and Hayes (88) have since demon- 

 strated that the paternal chromosome migrates 

 during fertilization in an orderly, progressive 

 way. When fertilization is prematurely inter- 

 rupted, the chromosome may be broken so that 

 only anterior markers appear among the re- 

 combinants. All of the genetic markers are 

 arranged in a single linkage group and their 

 order can be established either by timing their 

 passage during fertilization or by their statisti- 

 cal association with one another among the re- 

 combinants. Finally, the transfer of genetic 

 markers can be correlated with the transfer of 



DNA as inferred from the lethal effect of the 

 radioactive decay of incorporated P 82 (27). 



Sexual recombination is one of the methods 

 for analyzing the gene-enzyme relationship. 

 The studies so far are fragmentary but they 

 support the conception that the gene is a string 

 of nucleotides which must function as a co- 

 herent unit in order to produce an active en- 

 zyme (4, 33, 67, 15, 90). However, metabolic 

 blocks may originate through interference 

 with accessory regulatory mechanisms instead 

 of the fundamental capacity to produce the 

 enzyme. For example, many "lactase-nega- 

 tive" mutants have an altered pattern of en- 

 zyme induction or a defective permease system 

 for substrate transport (55, 65). Several labora- 

 tories are now working to correlate the relative 

 sequence of genetic defects with the sequence 

 of corresponding alterations in enzyme pro- 

 teins; this may be the next best approach to the 

 coding problem short of a system where a pure 

 DNA can be matched with its protein pheno- 

 type. 



At first these recombination experiments 

 were confined to a single strain of E. coli, K-12. 

 For many purposes this is a favorable choice 

 of material — perhaps the main advantage is 

 the accumulation of a library of many thou- 

 sands of substrains carrying the various mark- 

 ers called for by the design of genetic tests. 

 However, strain K-12 is rather unsuitable for 

 serological studies, having lost the character- 

 istic surface antigens which are the basis of 

 serological typing. In any event it would be 

 important to know the breeding structure of 

 the group of enteric bacteria. Systematic 

 studies have therefore been made of the inter- 

 fertility of different strains of bacteria, princi- 

 pally with a convenient tester of the K-12 strain 

 (39, 93). About one-fourth of the serotype 

 strains of E. coli are fertile with strain K-12, 

 and in at least some instances with one another. 

 Whether the remaining three-fourths of strains 

 are completely sterile, or whether they include 

 different, closed, breeding groups (i.e., differ- 

 ent genetic species) has not been systematically 

 tested, partly because of the preliminary work 

 needed to establish suitable strains. 



E. coli K-12 is also interfertile with a num- 

 ber of strains of Shigella spp. (59). Finally al- 

 though attempted crosses of E. coli with many 



104 



