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 lon mutant. Analysis of the determined nucleotide sequence of the lon 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 94, 59, and 54% 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 lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon 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 lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon 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.
Department of Microbiology and Immunology, University of Leicester, United Kingdom.
J A
JA
Eastgate
N
N
Taylor
M J
MJ
Coleman
B
B
Healy
L
L
Thompson
I S
IS
Roberts
D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic / Q000235:genetics; D049070:Protease La; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La