Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm

Cecilia Bartoleschi, Maria Chiara Pardini, Claudia Scaringi, Maria Celeste Martino, Carlo Pazzani, Maria Lina Bernardini

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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.
Original languageEnglish
Pages (from-to)613 - 626
Number of pages14
JournalCellular Microbiology
Issue number9
Publication statusPublished - Sep 2002
Externally publishedYes


All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Virology

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