Interactions Of Soil Clay Minerals With CSF Pheromone And Effects On Natural Transformation Of Bacillus Subtilis

In this study, we chose a Gram-positive bacterial Bacillus subtilis, which was an indigenous microorganism and abounded in earth; three widespread minerals: montmorillonite, kaolinite and goethite; and the CSF pheromone, which is an important regulator for B. subtilis forming competence as study materials. And the interaction between the CSF pheromone and three widespread minerals (montmorillonite, kaolinite and goethite) and how these interactions affect the B. subtilis nature transformation were studied. Besides, the plasmid pHTG was constructed to detect the frequency of transformation. The main results were listed as follows:1. The recombinant plasmid pHTG was used to detect the transform capacity of B.subtilis by Ery antibiotic resistance gene and GFP gene. It was constructed by inserting strong promoter p43 and gfpmut3a which can be high-efficiency expression in B. subtilis, and then can emerge a stable green fluorescence in B. subtilis. So when B. subtilis can grow in selection medium containing Ery antibiotic and can be detected green fluorescence by fluorescence microscopy, the plasmid pHTG was transformed into B. subtilis.The results show that the combination of green fluorescence and antibiotic was a reliable method to detect the transformation frequency.2. Adsorption of pHTG on the soil active particles. The trace DNA were mixed with the three clay minerals solutions to clarify the adsorption characteristic of pHTG on permanent-charge soil active particles. The maximum amount of pHTG adsorbed followed the order:goethite> kaolinite> montmorillonite. The pHTG adsorption was found a slow decrease with the increasing pH from 5.0 to 8.0. No remarkable change of adsorption was observed with increasing of temperature from 15 ℃ to 37 ℃. Electrolyte can promote pHTG adsorption on minerals and divalent ion was more powerful than monovalent ion in this process.3. HPLC and microcalorimetry were used to clarify the adsorption-desorption characteristic of CSF pheromone on montmorillonite, kaolinite and goethite. The adsorption isotherms of CSF pheromone was conformed to the Langmuir equation (R2> 0.96). The maximum amount of CSF pheromone adsorbed was in the order:kaolinite> montmorillonite> goethite. Adsorption process was an exothermic reaction, the amount of CSF pheromone adsorption decreased when temperature increased from 20 ℃ to 40 ℃. Mg2+ was more efficient than K+ in promoting CSF pheromone adsorption on three minerals. Desorption of adsorbed CSF pheromone on montmorillonite, kaolinite and goethite in Tris-HCl and ddH2O ranged from 75.3%～81.84%,80.44%～92.47%, 92.75%～94.99% respectively. From it we can known that montmorillonite was more strong affinitive.The bioactivity of adsorbed CSF pheromone was in the order:kaolinite> montmorillonite> goethite. This results can help us to understand the fate of CSF and how environmental impact of this process.4. Effect of different minerals and different mineral concentration on B. subtilis natural transformation was studied. As the above adsorption results, different minerals has different influence on this process, montmorillonite was the most and goethite was the lest. What’s more, with the increase of mineral concentration, the transformation efficiency was decreased. When mineral concentration increased from 0 mg/mL to 8 mg/mL, the transformation efficiency reduce two orders of magnitude and montmorillonite reduce more than other two minerals.This results can help us to understander the fate of CSF pheromone in environment and the effect of extraneous factors on transformation of B. subtilis.