Characterisation of phagosensor constructs.

Abstract

<p>(A) Three PCR fragments containing the <i>gfpmut2</i> gene under the control of either P<i>rrnB</i>, P<i>rplU</i> or P<i>bolA</i> promoter (red boxes), the kanamycin resistance-cassette and 80 bp homologous to the insertion region (dark and light blue) were recombined into the <i>hkaO</i>/<i>hkaP</i>, <i>hkaP</i>/<i>hkaQ</i> or <i>hkcE</i>/<i>hkcF</i> intergenic regions on the HK620 genome by the lambda Red recombination. (B) Fluorescence intensities of infected bacterial population measured by flow cytometry. Bacteria were infected for 1 hour at 37°C. Cytometry histograms represent the number of fluorescent or non fluorescent cells gated to the bacterial population as a function of the fluorescence intensity. The dotted line represents the limit between negative fluorescence (GFP-) and positive fluorescence (GFP+) determined by the fluorescent negative control WT HK620. Color code: non infected <i>E</i>. <i>coli</i> TD2158 (red), <i>E</i>. <i>coli</i> TD2158 infected with WT HK620 (orange) or recombinant phages HK620::P<i>bolA</i>-<i>gfp</i> (purple), HK620::P<i>rplU</i>-<i>gfp</i> (green), HK620::P<i>rrnB</i>-<i>gfp</i> (blue) (C) Histograms represent the means of median fluorescence intensities obtained with triplicate samples for 3 constructs. Color code is identical to (B).</p