Stimulation Of RP4 Conjugative Transfer By Nano-alumina

Conjugative transfer of self-transmissible plasmid in environment plays a crucial role in the dissemination of antibiotic resistance genes among bacterial populations. These bacteria might spread via the food chain to humans and intensify drug resistant problem. Expression of conjugative gene is tightly regulated, thus conjugative transfer frequency is affected by a variety of factors. Nanomaterials are increasingly utilized and will be inevitable released into soils and waters. Due to their unusual physicochemical properties such as small size and large surface area, nanomaterials are highly active and might influence plasmid conjugative transfer, but there is no such report yet at present. So we studied the effects of nano-alumina on RP4 conjugative transfer and then explored the mechanism by morphology, biochemistry, molecular biology and proteomics approaches.At first the effects of nano–alumina on RP4 conjugative transfer in terms of the variation in the number of transconjugants formed was investigated. Mating was performed in saline at 25℃without stirring for 8h with the initial density of parent cell were on the order of 109 cfu/ml with donor-to-recipient ratio fixed at 1:3. 5mmol/L and 50mmol/L nano-alumina could stimulate RP4 conjugative transfer by 150-fold and 40-fold respectively , with was proved by TEM micrographs that donor and receptor cell treated by 5mmol/L and 50mmol/L nano-alumina were prone to from conjugational junctions, but which was rarely seen in control mixture.Then we studied the effect of mating time, density of parent cell, pH and temperature on plasmid transfer in the presence of different concentrations of nano-alumina. The number of transconjugants increased as we prolonged mating time. When the initiate density of parent cell was on the order of 105cfu/ml, 5mmol/L and 50mmol/L nano-alumina inhibited conjugation completely while 0.5mmol/L nano-alumina stimulated conjugation most predominantly (P<0.001). As the initiate parent cell varied from 106cfu/ml to 108cfu/ml, the effect of nano-alumina depended on cell density and concentration of material.5mmol/L nano-alumina could increase RP4 conjugative transfer by approximately 100 to 400-fold respectively (P<0.001), while 50mmol/L Al2O3 nanoparticle could only enhance RP4 conjugative transfer when initiate parent cell were on the order of 108cfu/mL(P<0.001) and 0.5mmol/L nano-alumina could activate RP4 conjugative transfer as parent cell density were at the range of 106 to 107cfu/ml (P<0.05). Variations in temperature between 15℃to 37℃, as well as in pH between 6.5 and 8.0 did not significantly affect the number of transconjugants.In the third part LSCM and TEM were utilized to obtain information about the spatial distribution of nanoparticles and the morhpological change of parent cells. LSCM images showed that more nano-alumina combined with cells treated with 50mmol/L than 5mmol/L group, and nano-alumina could penetrate inside bacteria and caused considerable changes in the cell membranes,as illustrated by TEM micrographs. Many cells had lost the distinctiveness of the cell membrane upon treatment by 50mmol/L nano-alumina. Oxidative stress induced by nano-alumina might explain the reason for morhpological change, upon treatment by 5 mmol/L and 50mmol/L nano-alumina, total antioxidant capacity (T-AOC) of bacteria increased, with activities of the corresponding anti-oxidation enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) increased as well.To study the effect of nano-alumina on transcription of RP4 transfer gene, trbBp-lacZ and trfAp-lacZ transcriptional fusions were constructed and thenβ-calactosidase activity were assayed to measure the activity of trbBp and trfAp, which are the key promoters of Mpf system and Dtr system respectively. Comparation of lacZ activity showed that after treated by 5mmol/L and 50mmol/L nano-alumina, trbBp and trfAp activity were both significantly increased, while the difference between 5 mmol/L and 50mmol/L group was not significant.TrbBp and trfAp are regulated by global regulatory factor korA, korB and trbA. Real-time quantitative PCR assay was designed for detection and quantification of global regulatory factor korA, korB and trbA transcripts in total RNA isolated from conjugation mixture. The result showed that korB and trbA mRNA expression of mating cell treated by 5mmol/L and 50mmol/L nano-alumina were increased significantly compared with control group.Transcript of korA mRNA increased too but with no significant difference. Transcription of korA, korB and trbA between 5 mmol/L and 50mmol/L nano-alumina group was not significant .To screen the candidate genes that responded to 5mmol/L nano-alumina, the protein expression patterns of mating cell was examined two-dimensional polyacrylamide gel electrophoresis(2-D PAGE), then three differentially expressed proteins in cell treated by 5mmol/L nano-alumina were sucessfully identified with matrix assisted laser desorption/ionization-time of fight (MALDI-TOF) mass spectrometer, among which two protein identified were malate synthase G and Pyruvate kinase respectively, both were highly expressed by the donor cell,the other was a probable peroxidase, expressed by recipient.According to the results we could draw the following conclusions:1. Nano-alumina can improve RP4-mediated conjugative transfer and the promoting effect of nano-alumina varies with concentration of nano-alumina and density of mating cell. As the donor and recipient in conjugation mixture decreased, the concentration of nano-Al2O3 that can stimulate RP4 conjugative transfer maximumly reduces too.2. Nano-alumina can generate reactive oxygen species, attack and cause considerable changes in the cell membranes. The morphological change.of mating cells will be intensified as concentration of nano-alumina increased. Slight damage of cell membrane facilitates RP4 conjugative transfer, but conjugation will be inhibited if the cell membrane is extensively disrupted, which might be the reason why conjugative transfer of RP4 decreases as concentration of nano-alumina increase under many conditions.3. Nano-alumina might stimulate the transcription of global regulator factor korB and trbA directly to regulate the expression of conjugative transfer genes, which lead to the increasing transcriptional activity of trbBp and trfAp in mating mixture, and facilitate conjugative transfer of RP4 .4. In response to increasing expression of RP4 transfer genes, the donor cell produce more energy metabolism related enzymes upon treatment by 5mmol/L nano-alumina. And a probable peroxidase are highly expressed by recipient cell, which might involved in detoxification of reactive oxygen species induced by nano-alumina.To sum up, the present work was the first to explore the promoting effect of nano-alumina on RP4 conjugative transfer and its mechanism was studies as well. The research reminds us as a new environment contaminant, nano-material might hazard water environment and human health, which provides a theoretical basis for prevention of nano-materials.