We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a Jibrary of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels (<10 μg ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to, induce a cytophatic effect - plaque - on a cell mono-layer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell mono-layer in the presence of Cm and give a positive result in the plaque assay. Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) β-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encoding unknown functions were also, traped and selected by this new IVET-based protocol.
All Science Journal Classification (ASJC) codes
Bartoleschi, C., Pardini, M. C., Scaringi, C., Martino, M. C., Pazzani, C., & Bernardini, M. L. (2002). Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm. Cellular Microbiology, 4(9), 613 - 626. https://doi.org/10.1046/j.1462-5822.2002.00216.x