Abstract
Infection of bacteria with deoxyribonucleic acid (DNA) extracted from bacterio phages, that is called “transfection”, was found in Mycobacterium smegmatis in 1963. Evidence for showing that DNA but not any phage particles contaminated in the DNA samples produces infective centers was presented as follows: (1) the in fectivity was destroyed completely by catalytic amounts of DNase, (2) phage antiserum did not reduce number of plaque-forming units, (3) Tween 80, which prevents, phage adsorption, did not prevent the infection, (4) a phage-resistant mutant of the host was infected by the sample, (5) host cells in a late log phase of the growth were competent for the infection (Fig. 2).
Since it was known in Bacillus subtilis, Pneumococcus and Haemophilus that cells, competent for transfection are also competent for genetic transformation, the phenomenon of transfection found in Mycobacteria has been widely studied by many investigators from the standpoint of genetic transformation. For instance, we found that glycine has capacity to sensitize mycobacterial cells not only for the induction of cell lysis by lysozyme but also for the transfection (Fig. 3) These observations were expanded from glycine to various d-type amino acids. Based on these knowledges, we devised a new, gentle method for isolating DNA from amino acid-sensitized mycobacteria with lysozyme and phenol (Fig. 4). Employing the amino acid-sensitized cells competent for transfection and DNA isolated gently from various strains of Mycobacteria, experiments aiming at genetic transformation. were carried out. However all attempts have been ended in negative results.
Apparently, mycobacterial cells have ability to uptake DNA and have dark repair system for UV-damaged DNA. As described later, they also have intracellular mechanisms of recombinating bacterial DNA with their chromosomes. The reason why mycobacterial cells competent for transfection can not be transformed with bacterial DNA has not yet been clear; we assume that they might lack ability to recover after possible DNA uptake and recombinative events.
Transduction with mycobacteriophage was reported by Ramakrishnan and his coworkers using phage 13 and M. smegmatis strain SN2. We confirmed their results and added some positive data using additional genetic markers (Table 2). Attempts for obtaining transducing phages other than phage 13, however, were all failed.
In 1970, genetic recombination was demonstrated in M. smegmatis. Crosses between nutritionally complementary auxotrophs derived from one strain were infer tile whereas those from different strains formed recombinants (Fig. 6, Table 4 and 5). Conjugation rather than transformat ion and transduction was suggested as the gene transfer mechanism. Backcrosses of recombinants by either parental strain indicated four different types of mating behavior, suggesting that the mycobacterial compatibilities are controlled by at least two different factors (Table 7).
Sexual conjugation rather than cell fusion was proposed as the zygote-formation mechanism, based on the following facts: (1) analysis of segregation of unselected markers in recombinant progeny obtained in various cross systems revealed that one particular parent contributes the majority of alleles in nearly all of the recombinants, (2) mating medium containing streptomycin (SM) prevented recombination when one particular parent was SM resistant and another was sensitive, but it did not prevent recombination when the former parent was SM-sensitive and the latter was resistant (Table 6), and (3) crosses were infertile when one particular parent was recombination deficient mutant (rec-) and another was not (rec+), but crosses were fertile when the former strain was rec+ and the latter was rec-(unpublished data).
