Study of Molecular Mechanisms of Escherichia coli Response to Monochloramine Using DNA Microarray Technology and Flow Cytometry

Resistance to disinfectants may be partially responsible for the higher than expected persistence of certain bacteria in drinking water distribution systems. This resistance could be due to environmental factors, such as exposure to lower than anticipated disinfectant concentrations because of mass transfer resistance when bacteria are present in biofilms. Alternatively, the composition of proteins within bacterial cells may change when the cells are exposed to disinfectants, as part of a specific response to that disinfectant. Inactivation curves with stationary phase Escherichia coli, using high (3.0-5.0 mg/L as Cl2) and low (0.3-0.5 mg/L as Cl2) concentrations of monochloramine, showed similar inactivation efficiency using a delayed Chick-Watson model equation. Initial DNA microarray experiments identified four genes (ykgB, ykgC, ykgI, and ykgL) with high inductions (around 20-fold induction) after monochloramine treatment to be in the same region of the E. coli K-12 chromosome. The function of these genes is currently unknown. Flow cytometric analysis of the initial effects of exposure to monochloramine on E. coli mutants lacking three of these genes (ΔykgB, ΔykgI and ΔykgL) indicated that when stained with propidium iodide, two of the mutants had different patterns of fluorescent dye incorporation, compared to the wild type cells. Propidium iodide, which can only enter cells with damaged cell walls, was incorporated at higher rates in mutants ΔykgB and ΔykgL than in the other mutant ΔykgI or in the wild type strain. DiCO3, a membrane potential dye, was used to stain the wild type and the mutants lacking proteins associated with the cell wall. Mutant ΔykgL (outer membrane protein) exhibited exaggeratedly hyperpolarized membranes compared to the wild type when treated with 4 mg/L monochloramine as Cl2, while untreated (control) ΔykgL cells had lower polarization levels than comparable wild type or untreated cells lacking ykgB (cytoplasm membrane protein). In contrast, the untreated mutant ΔykgB consisted of two populations: one had comparable levels of membrane polarization to the wild type cells the other was depolarized. Our results demonstrate that DNA microarray technology and Flow Cytometry allow us to unravel molecular mechanisms of bacterial resistance to disinfectants.