Physiological factors involved in the transformation of Mycobacterium smegmatis

Abstract

Transfer of streptomycin resistance and changes from methionine and leucine auxotrophy to prototrophy were achieved in Mycobacterium smegmatis by transformation. Recipient cells were more resistant to mitomycin C and methyl methanesulfonate treatments than were wild-type cells. A high level of calcium ions was essential for transformation, especially during DNA adsorption, whereas the presence of magnesium ions and the exposure of recipient cells to mild doses of UV light enhanced recombination frequencies. Transformants were not isolated when recipient cell-DNA mixtures were first treated with deoxyribonuclease. Recipient cells at various stages of growth showed similar transformabilities. Transformation was successful only when recipient cells were incubated on rich agar medium after mixture with DNA. Exposure of recipient cells to Pronase before treatment with donor DNA did not affect transformation, suggesting the absence of a protein competence factor. Throughout the present experiments, cotransformation frequencies were very low and unselected-marker segregation patterns were independent, indicating that the methionine, leucine, and streptomycin markers are not closely linked in M. smegmatis. Despite the medical importance of mycobacteria, knowledge of genetic recombination in this genus remains limited. Experimental procedures in transduction (12, 13, 20-22, 41) and conjugation (30, 32, 40, 44) have recently been established for various species of mycobacteria, but results of transformation experiments have been inconclusive (4, 14, 43). Transformation in mycobacteria was first reported by Katunuma and Nakasato (23), who claimed that streptomycin resistance was transferred to Mycobacterium avium through DNA extracted from streptomycin-resistant strains. Tsukamura et al. (48) reported the transfer of isoniazid and streptomycin resistance to M. avium after exposure for 5 days to high concentrations of DNA extracted from resistant strains. Gelbart and Juhasz (12) and Juhasz et al. (S. E. Juhasz, S. M. Gelbart, and L. DeSalle, Bacteriol. Proc., p. 35, 1971) described xylose utilization transferred to Mycobacterium phlei through transformation. In contrast to these observations, Bloch et al. (3) failed to transfer streptomycin and isoniazid resistance to M. phlei, M. smegmatis, M. bovis, and M. tuberculosis, using purified DNA from respective resistant strains. They were also unsuccessful in transferring the ability to synthesize pigments to M. smegmnatis from M. phlei or the virulence of M. tuberculosis H37Rv to the avirulent M. tuberculosis H37Ra through transformation. Similarly, Bradley (4) was unable to transfer isoniazid, streptomycin, or cycloserine resistance or mycobacteriophage susceptibility to M. smegmatis, M. kansasii, M. bovis, M. tuberculosis, and M. intracellulare through

Genomic Signatures of Distributive Conjugal Transfer among Mycobacteria

“…Mycobacterium canettii encodes the genetic elements known to be required for DCT: ESX-1, tad , and lpqM ( Flint et al 2004 ; Coros et al 2008 ; Nguyen et al 2009 ), making this mechanism plausible. Natural transformation has been observed in M. smegmatis and M. avium ( Tsukamura et al 1960 ; Norgard and Imaeda 1978 ) and could be an alternative mechanism of LGT in M. canettii . Little is known about the underlying mechanism or genetic requirements for natural transformation in mycobacteria.…”

Bacterial gene transfer by natural genetic transformation in the environment.

“…(94). In Agmenellum quadruplicatum (341), Deinococcus (Micrococcus) radiodurans, and Mycobacterium smegmatis (250,365) competence proceeds throughout exponential growth and declines during the stationary phase. The portion of cells able to take up DNA in a culture can be estimated from the transformation frequency obtained, e.g., with an auxotrophic marker on homologous chromosomal DNA, considering that the marker is present on only 1 of approximately 100 to 200 DNA fragments per chromosome.…”

Pathogenic Determinants of the Mycobacterium kansasii Complex: An Unsuspected Role for Distributive Conjugal Transfer

“…However, HGTs occur widely in non-tuberculous mycobacteria. Natural transformation, characterized by the horizontal acquisition of usually small DNA fragments (3-5 Kb) by an active bacterial uptake, was reported in M. smegmatis and M. avium [26][27][28]. Transduction, mediated by bacteriophages, also occurs in the Mycobacterium genus [29,30].…”