Dual Labeling of Pseudomonas putida with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies

The ionic liquid 1-alkyl-3-methylimidazolium demonstrates comparable antimicrobial and antibiofilm behavior to a cationic surfactant

<div><p>Biofilms are problematic in health and industry because they are resistant to various antimicrobial treatments. Ionic liquids are a novel class of low temperature liquid salts consisting of discrete anions and cations, and have attracted considerable interest as safer alternatives to organic solvents. Ionic liquids have interesting antimicrobial properties and some could find use in the development of novel antiseptics, biocides and antifouling agents. The antimicrobial and antibiofilm activity of 1-dodecyl-3-methylimiazolium iodide ([C₁₂MIM]I) was studied using the clinically important bacterial pathogens, <i>Staphylococcus aureus</i> SAV329 and <i>Pseudomonas aeruginosa</i> PAO1. The ionic liquid increased cell membrane permeability in both <i>S. aureus</i> and <i>P. aeruginosa</i> cells and impaired their growth, attachment and biofilm development. The ionic liquid exhibited superior antimicrobial and antibiofilm activity against the Gram-positive <i>S. aureus</i> compared to the Gram-negative <i>P. aeruginosa</i> cells. <i>Bac</i>Light™ staining and confocal microscope imaging confirmed that the ionic liquid treatment increased the cell membrane permeability of both the Gram-positive and Gram-negative bacteria. In addition, the antimicrobial and antibiofilm properties of [C₁₂MIM]I were similar or superior to those of cetyltrimethylammonium bromide (CTAB), a well-known cationic surfactant. It is concluded that the ionic liquid induced damage to bacterial cells by disrupting cell membrane, leading to inhibition of growth and biofilm formation. Overall, the results indicate that the ionic liquid 1-dodecyl-3-methylimiazolium iodide was effective in preventing <i>S. aureus</i> and <i>P. aeruginosa</i> biofilms and could have applications in the control of bacterial biofilms.</p> </div