Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora.
Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a lambda-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.
Hinton
J C
JC
Department of Biological Sciences, University of Warwick, Coventry, UK.
Salmond
G P
GP
D000469:Alkaline Phosphatase / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D004251:DNA Transposable Elements; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation
D004251:DNA Transposable Elements; D006867:Hydrolases; D000469:Alkaline Phosphatase