Excision of a P4-like cryptic prophage leads to Alp protease expression in Escherichia coli

Abstract

The Escherichia coli K-12 alpA gene product, when overproduced from a multicopy plasmid, leads to suppression of the capsule overproduction and UV sensitivity phenotypes of cells mutant for the Lon ATP-dependent protease. This suppression has previously been shown to correlate with increased in vivo activity of a previously unknown energy-dependent proteolytic activity capable of degrading Lon substrates, the Alp protease. We show in an accompanying paper that alpA, which has homology to a short open reading frame in bacteriophage P4, acts as a positive transcriptional regulator of slpA, a gene linked to alpA and necessary for suppression of lon mutants (J. E. Trempy, J. E. Kirby, and S. Gottesman, J. Bacteriol. 176:2061-2067). The sequence of slpA suggests that it encodes an integrase gene closely related to P4 int and that both alpA and slpA are part of a cryptic P4-like prophage. AlpA expression increases SlpA synthesis. Increased SlpA leads, in turn, to the excision and loss of the cryptic prophage. Excision is dependent on integration host factor as well as on SlpA. Prophage excision is necessary but not sufficient for full expression of the Alp protease. A second function (named AHA) allows full protease expression; this function can be provided by the kanamycin resistance element from Tn903 when the element is present on a multicopy plasmid. Excision and loss of the cryptic prophage apparently allow expression of the Alp protease by inactivating a small stable RNA (10Sa RNA) encoded by the ssrA gene. The precursor of this RNA has its 3' end within the cryptic prophage; the mature 3' end lies within the prophage attL site. Inactivation of ssrA by insertional mutagenesis is sufficient to allow expression of the suppressing Alp protease, even in the presence of the cryptic prophage. Therefore, 10Sa RNA acts as a negative regulator of protease synthesis or activity, and prophage excision must inactivate this inhibitory function of the RNA.