Effects of Electroporation on Antibiotic Susceptibility and Adhesive Activity to n -Hexadecane in Rhodococcus ruber IEGM
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cts of Electroporation on Antibiotic Susceptibility and Adhesive Activity to n-Hexadecane in Rhodococcus ruber IEGM 231 M. S. Kuyukinaa, b, *, A. M. Varushkinaa, b, and I. B. Ivshinaa, b aInstitute
of Ecology and Genetics of Microorganisms, Perm Federal Research Center, Ural Branch, Russian Academy of Sciences, Perm, 614081 Russia b Perm State University, Perm, 614990 Russia *e-mail: [email protected] Received April 30, 2020; revised June 12, 2020; accepted July 2, 2020
Abstract—The effects of electroporation on the viability, antibiotic susceptibility, and adhesive activity of Rhodococcus ruber IEGM 231 towards n-hexadecane were studied. An 85–96% reduction in cell viability was observed after electroporation at 9 and 12.5 kV/cm. As a result of electroporation, the antibiotic susceptibility of rhodococci to benzylpenicillin, gentamicin, clotrimazole, neomycin, and cefazolin increased by 8–46%. The dynamics of antibiotic susceptibility was found to differ depending on the antibiotic used and the cell recovery time after electroporation. The highest antibiotic susceptibility, which was correlated with an increase in adhesion to hydrocarbon, was recorded for electroporated cells after a 24-h recovery period, which corresponded to the beginning of the stationary growth phase. However, spontaneous mutants of R. ruber IEGM 231 resistant to 200 μg/mL of kanamycin were characterized by reduced cell-wall hydrophobicity. These findings can be used for the electrotransformation of hydrocarbon-oxidizing rhodococci in order to increase the efficiency of recombinant clone selection via a decrease in the cell recovery period and the antibiotic concentration in selective medium. Keywords: actinobacteria, Rhodococcus, electroporation, antibiotic susceptibility, hydrophobicity, cell adhesion DOI: 10.1134/S0003683820060083
INTRODUCTION Electroporation is the creation of pores in a cell membrane under the influence of a short-term, electrical impulse of high voltage. It is widely used in medicine and is a convenient tool for biotechnological purposes, such as the genetic transformation of cells, the extraction of biomolecules, the inactivation of microorganisms in wastewater, the nonthermal pasteurization of food, etc. [1]. Water dipoles penetrate the lipid bilayer of the membrane under the influence of a short pulse with a duration of 10 μs to 10 ms and a strength of 1 to 20 kV/cm and reorient the phospholipids, forming pores for the transport of macromolecules both inside and outside the cell [2]. A high-voltage electrical impulse also affects the bacterial cell wall; under its influence, numerous perforations of the peptidoglycan layer form and facilitate the transport of molecules [3]. The connection between the action of electric current on bacterial cells and the increase in their sensitivity to antibiotics, biocides, and solvents was proven. It is called the “bioelectric effect” [4]. The bioelectric effect has been studied under the influence of a constant weak electric current, but the effect of a high-
voltage current on th
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