CLONING, EXPRESSION, AND CHARACTERIZATION OF THE LON GENE OF ERWINIA-AMYLOVORA - EVIDENCE FOR A HEAT-SHOCK RESPONSE

The gene encoding the Lon protease of Erwinia amylovora has been clone d by complementation of an Escherichia coli ion mutant, Analysis of th e determined nucleotide sequence of the ion gene revealed extensive ho mology to the nucleotide sequences of cloned lon genes from E. coli, M yxococcus xanthus, and Bacillus brevis. The predicted amino acid seque nce of the E. amylovora Lon protease was 93, 59, and 53% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively, The -10 and -35 promoter region s of the cloned ion gene had extensive homology to the respective cons ensus sequences of E. coli heat shock promoters. Promoter mapping of t he ion gene located the start site 7 bases downstream of the -10 regio n. Cloning of the ion promoter upstream of a cat reporter gene demonst rated that expression of the E. amylovora Ion gene was inducible by a heat shock This is the first demonstration of a heat shock-regulated g ene in E. amylovora. Site-directed mutagenesis of the -10 region of th e lon promoter confirmed that the heat shock expression of the E. amyl ovora ion gene may. be mediated by a sigma(32)-like factor. Insertiona l inactivation of the E. amylovora chromosomal ion gene confirmed that the ion gene was not essential for either vegetative growth or infect ion of apple seedlings, E. amylovora ion mutants had increased sensiti vity to UV irradiation and elevated levels of extracellular polysaccha ride, suggesting comparable roles for the Lon proteases in both E. amy lovora and E. coli.