Can Ca2+-dependent competence be repeatedly induced in the same Escherichia coli cells?

With the help of devised multicycle consecutive transformation (MCT) it is shown that Ca2+-dependent competence can be repeatedly induced in the same population of Escherichia coli cells. The same fraction of cells is induced to competence and transformed during MCT. In contrast to the results on classical transformation with mixed DNA preparations, no double transformants are observed in MCT. The competent cells and transformants are found to be more fragile than nontransformed cells. The latter are represented presumably by the cells that have not absorbed exogenous plasmid DNA. The results suggest that there is strong interference between plasmid DNAs during MCT, and that the presence of exogenous DNA makes the cells more sensitive to the apparently harmful procedure of repeated competence induction.

[Auxotrophic mutations formed on the incorporation into the E. coli C600 chromosome of transposon Tnl that determines ampicillin resistance]. / Mutatsii auksotrofnosti, voznikaiushchie pri vstraivanii v khromosomu E. coli C600 transpozona Tnl, opredeliaiushchego ustoichivost' k ampitsillinu.

Incorporation of transposone Tnl of ampicillin resistance into the chromosome of E. coli C600 resulted in formation of at least 8 types of auxotrophic mutants. The transposone incorporates mainly into the loci the damage of which induces proline deficit (20 per cent). The sites of incorporation of transposone Tnl in the chromosomes of E. coli C600 and E. coli JC411 did not coincide.

[2 types of auxotrophic mutants occurring by insertion of the ampicillin transposon into the chromosome of E. coli K-12]. / Dva tipa auksotrofnykh mutantov, voznikaiushchikh pri vstraivanii ampitsillinovogo transpozona v khromosomu E. coli K-12.

Insertion of transpozone TnI determining ampicillin resistance into the E. coli K-12 chromosome resulted in formation of auxothrophic mutants of 2 types. The mutants of the first type carried thermosensitive mutation resulting in auxotrophy with respect to isoleucine at a temperature of 43 degrees C. Such mutants occurred with high frequency (up to 14 per cent with respect to the number of the survived cells with the chromosomes carrying inserted TnI) and had capacity for reversion to the phenotype of the wild type. The mutants of the second type occurred with a frequency 20--180 times lower than that of the mutants of the first type and did not reverse to the phenotype of the parent bacteria. It was found that the chromosome of E. coli K-12 possessed at least 7 sites available for transpozone TnI insertion.