Transformation and survival of donor, recipient, and transformants of Bacillus subtilis in vitro and in soil

The survival of Bacillus subtilis strains BD1512 and BD170 and the transfer by transformation of chromosomal genes coding for the synthesis of amino acids (threonine, tryptophan, histidine, leucine and methionine) and of a plasmid-borne gene coding for resistance to chloramphenicol in vitro and in sterile and non-sterile soil (the soil was amended to 3, 6, 9, or 12% (vol/vol) with the clay mineral, montmorillonite) were studied, as was the effect of water content on the transformation of chloramphenicol resistance in non-sterile soil. The survival of the vegetative cells and spores of the donor, recipient and three transformants (H1: a single-gene (histidine) transformant; HM2: a two-gene (histidine and methionine) transformant; and HLM3: a three-gene (histidine, leucine and methionine) transformant) was similar in vitro and in soil. In vitro, the numbers of cells of donor, recipient, and transformants decreased by approximately 1 to 2.1 orders of magnitude during a 14-d incubation, whereas the numbers of spores, which initially were about 2 to 3.2 orders of magnitude lower than the numbers of cells, increased so that the numbers of spores and cells on day 14 differed by only 0 to 0.2 orders. In sterile soil, the numbers of cells decreased by approximately 0.8 to 2 orders of magnitude, whereas the numbers of spores increased by approximately 0.6 to 2.5 orders during the 14-d incubation. Survival in non-sterile soil could not be determined because of the resistance of indigenous soil bacteria and fungi to chloramphenicol and cycloheximide, respectively, at the concentrations used. The transformation frequency during 6 d in vitro ranged from 1.2±0.40×10−7 to 7.9±0.65×10−6 for one gene; from 8.0±0.40×10−8 to 4.0±0.83×10−7 for two genes; and from 2.8±0.20×10−8 to 1.4±0.19×10−7 for three genes. In sterile soil, the transformation frequency during 6 d ranged from 6.5±0.30×10−8 to 1.8±0.10×10−6 for one gene; from 2.8±0.12×10−8 to 1.7±0.20×10−6 for two genes; and from 2.4±0.60×10−8 to 9.2±3.90×10−8 for three genes. The transformation frequency of one, two or three genes generally increased as the amount of montmorillonite added to the soil was increased. The frequency of transformation of chloramphenicol resistance in non-saturated (33 kPa water tension), non-sterile soil ranged from 4.3±0.12×10−7 to 2.0±0.54×10−6, whereas the frequency in water saturated, non-sterile soil ranged from 5.8±0.15×10−8 to 4.2±0.00×10−7. These studies showed that montmorillonite enhanced survival of the Bacillus strains and transformation of both chromosomal and plasmid-borne genes and that the optimal water content of soil results in optimal horizontal genetic exchange by transformation. These results should be considered in risk assessments of the release of genetically modified microorganisms to soil.