Oral Presentation The 2nd Prato Conference on the Pathogenesis of Bacterial Diseases of Animals 2012

Single-cell measurement of F1651 and Pap fimbrial phase variation in real-time (#17)

Rémi Lavoie 1 , Richard Graveline 1 , Josée Harel 1
  1. Universite de Montreal, Saint-Hyacinthe, QC, Canada

Background: Fimbriae F1651 (foo)synthesized by septicemic Escherichia coli strains belong to the type P (pap) family of adhesive factors and are under the control of a methylation-dependent transcriptional regulation, called phase variation. This mechanism arises from competition between two global regulatory proteins, Lrp and Dam, within the regulatory region and gives an heterogenous phenotype among a clonal population. If F1651 and Pap share similar regulatory mechanisms, F1651 expression however has its own specificity, showing a more heterogeneous phenotype, with a predominant ON state. Here, we adapted single cell measurement approaches to distinguish changes during phase variation of foo and pap operons. Methods and Results: To characterize thephase variation, we measured it using transcriptional fusions between the regulatory regions of pap and foo operons and the T7 RNA polymerase (T7 RNAP). Expression of T7 RNAP activates a specific promoter regulating the expression of gfp located on a plasmid. This enabled single cells in the ON and OFF states to be distinguished by flow cytometry and fluorescence microscopy. Flow cytometry confirmed previous analysis of phase variation  using lacZ reporter fusions but gave additional information. Each profile showed three distinct bacterial populations, corresponding to ON cells, OFF cells and intermediate level of fluorescence. While pap cells were mostly in the OFF state, foo cells showed a more heterogeneous phenotype. Similar patterns were observed when Pap and F1651 phase variations were monitored in real time on individual E. coli using fluorescence microscopy. Conclusion: Following single cells in real time, we have shown distinctive patterns of expression of the pap and foo operons. Moreover investigation of the behavior of single cells, confirmed Pap as a slow switcher and F1651 as a fast switcher. Therefore, the rapid fimbrial switch of F1651-ExPEC strains may enhance their adaptation to fluctuating environments and contribute their fitness.