Impact of nanoparticles on the Bacillus subtilis (3610) competence

International audience; Due to the physicochemical properties of nanoparticles, the use of nanomaterials increases every year in industrial and medical processes. At the same time, the increasing number of bacteria becoming resistant to many antibiotics, mostly by a horizontal gene transfer process, is a major public health concern. We herein report, for the first time, the role of nanoparticles in the physiological induction of horizontal gene transfer in bacteria. Besides the most well-known impacts of nanoparticles on bacteria, i.e. death or oxidative stress, two nanoparticles, n-ZnO and n-TiO 2 , significantly and oppositely impact the transformation efficiency of Bacillus subtilis in biofilm growth conditions, by modification of the physiological processes involved in the induction of competence, the first step of transformation. This effect is the consequence of a physiological adaptation rather than a physical cell injury: two oligopeptide ABC transporters, OppABCDF and AppDFABC, are differentially expressed in response to nanoparticles. Interestingly, a third tested nanoparticle, nAg , has no significant effect on competence in our experimental conditions. Overall, these results show that nanoparticles, by altering bacterial physiology and especially competence, may have profound influences in unsuspected areas, such as the dissemination of antibiotic resistance in bacteria. Due to the specific physicochemical properties of nanoparticles, e.g. high specific area, the use of nanomaterials increases every year in industrial and technological processes or in medical applications. Despite their exceptional qualities, their deleterious impact on the environment and health also gives rise to an increasing number of publications 1–4. Among their numerous uses, nanoparticles and particularly metal-oxide nanoparticles, have often been used for their antibacterial properties, reviewed in 5,6. They are also considered an alternative to antibiotic treatment because they can efficiently kill bacteria with no or a very limited emergence of drug resistance to date. Most often, the deleterious impacts of nanoparticles on cells and especially on bacteria are described as the result of a physical injury to cell integrity, either by disruption of the membrane and/or by the production of reactive oxygen species 5,7. Some recent publications have shown that nanoparticles can also impact internal physiological processes of bacteria such as stringent response, respiration, central metabolism, motility, sporulation or chromosome condensation 8–10. We have shown that exposure of the soil bacterium Bacillus subtilis (laboratory strain, 168) to n-TiO 2 and n-ZnO, under long-term adaptation growth conditions in a liquid medium, impacts its physiological state by modifying the central metabolism and stringent response 8. However, the natural biotope of Bacillus subtilis is the upper layer of soil, where it grows as a biofilm. It plays a role in rhizospheric processes as a symbiotic organism for plant 11. To mimic this physiological development and study the impact of nanoparticles during the formation of a biofilm in a contaminated soil, we studied the proteomic response of the ancestral strain Bacillus subtilis 3610, which is able to form a biofilm, contrary to the well known 168 laboratory strain. The bacteria were grown on soft agar plates containing n-ZnO and n-TiO 2. We show here that under these growth conditions, where the nanoparticles are not physically in contact with bacteria, n-TiO 2 and n-ZnO impact, in an opposite way, the expression of two oligopeptide ABC transporters, OppABCDF and AppABCDF, and consequently , the competence of Bacillus subtilis. The competence being one of first step of the transformation process, our results suggest that nanoparticles impact horizontal gene transfer in, at least Gram positive bacteria and may have significant impact on the appearance of antibiotic-resistant bacteria. Results Nanoparticles characterization. The sizes and aggregation states of the nanoparticles of n-TiO 2 and n-ZnO suspensions were characterized by DLS measurements 8 and TEM images analysis (sup data 1). The nominal size and morphological characteristics of n-TiO 2 and n-ZnO were observed by transmission electron micros-copy in H 2 O suspension. Supp data 1 shows representative images of each. The measured nominal sizes were