Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12

Escherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE), that encodes a haemolytic 34 kDa protein. Recombinant E, coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of C lyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the transla tional start point of the clyA gene and demonstrated that the clyA gene pro duct is not N-terminally processed during transport. The transcription of c lyA from its native promoter region was positively controlled by SlyA, a re gulatory protein found in E. coli, Salmonella typhimurium and other Enterob acteriaceae. SlyA-controlled transcription started predominantly 72 bp upst ream from clyA, as shown by primer extension. The corresponding putative pr omoter contains an unusual -10 sequence (TATGAAT) that is separated from a conventional -35 sequence by a GC-rich spacer. Site-directed deletion of th e G in the -10 sequence abrogated the SlyA requirement for strong ClyA prod uction, whereas a reduction in the G+C content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection ass ays and lipid bilayer experiments suggested that ClyA forms stable, moderat ely cation-selective transmembrane pores that have a diameter of about 2.5- 3 nm.