Publications by Year: 1994

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.

We have previously found that plasmids carrying the Escherichia coli alp gene (now to be called alpA) suppress two phenotypes of a delta lon protease mutant, overproduction of capsular polysaccharide and sensitivity to UV light. Suppression of these lon phenotypes is most likely explained by the increased degradation of the Lon substrates responsible for these phenotypes. We have called this suppressing protease activity Alp protease. The Alp protease activity is detected in cells after introduction of plasmids carrying the alpA gene, which encodes an open reading frame of 70 amino acids. Insertions which abolish Alp activity interrupt this open reading frame. We have used Tn10 and lambda placMu mutagenesis to identify a chromosomal locus, slpA, that is required for alpA+ suppression of delta lon. This locus maps at 57 min, close to the chromosomal location of alpA. The expression of beta-galactosidase from a lac transcriptional fusion to slpA is increased six- to eightfold when the alpA+ gene is present on a multicopy plasmid. Therefore, AlpA acts as a transcriptional regulator of the slpA gene(s); activation of slpA transcription is necessary to suppress the phenotypes of a delta lon mutation. In an accompanying paper (J. E. Kirby, J. E. Trempy, and S. Gottesman, J. Bacteriol. 176:2068-2081, 1994), we show that neither AlpA nor SlpA is a component of the protease itself but that they are part of a regulatory cascade which leads to expression of the Alp protease.