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 cloned by complementation of an Escherichia coli ion mutant, Analysis of the determined nucleotide sequence of the ion gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence 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 regions of the cloned ion gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the ion gene located the start site 7 bases downstream of the -10 region. Cloning of the ion promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora Ion gene was inducible by a heat shock This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora ion gene may. be mediated by a sigma(32)-like factor. Insertional inactivation of the E. amylovora chromosomal ion gene confirmed that the ion gene was not essential for either vegetative growth or infection of apple seedlings, E. amylovora ion mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.