Rossiter, Amanda Eve (2011)
Ph.D. thesis, University of Birmingham.
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Autotransporters represent a diverse family of virulence effectors that are secreted from Gram-negative bacteria by the Type V Secretion System. Their initial description coined the term ‘Autotransporter’ to embody the notion that their three-part architecture governs their navigation through the bacterial cell envelope. The Pet cytotoxic autotransporter is secreted by the diarrhoeal pathogen, Enteroaggregative Escherichia coli (EAEC) and was used as a model to study autotransporter biogenesis. Following a global transposon mutagenesis of EAEC, novel accessory factors were identified that are required for Pet biogenesis, including the transcription factors CRP and Fis, periplasmic chaperones and components of the β-barrel assembly machinery (BAM) complex. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation and show that this co-activation mechanism extends to functionally similar autotransporters. Furthermore, we highlight novel components of the BAM complex required for AT secretion. This work builds on previous studies that, in recent years, have challenged the ‘auto’ nature of this secretion process causing a paradigm shift towards a much more complex mechanism of AT secretion than initially suggested.
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