Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

Abstract

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.

• Authors: M R Maurizi, P Trisler, S Gottesman
• Journal: Journal of bacteriology (J Bacteriol) Vol. 164 Issue 3 Pg. 1124-35 (Dec 1985) ISSN: 0021-9193 [Print] United States
• PMID: 2999072 (Publication Type: Journal Article)

Chemical References

• DNA Transposable Elements
• Puromycin
• Adenosine Triphosphate
• DNA Restriction Enzymes
• Peptide Hydrolases

Topics

• Adenosine Triphosphate (metabolism)
• Amino Acid Sequence
• Bacteriophage lambda (genetics)
• Base Sequence
• Chromosome Deletion
• Cross Reactions
• DNA Restriction Enzymes (metabolism)
• DNA Transposable Elements
• Electrophoresis, Agar Gel
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (genetics)
• Mutation
• Peptide Hydrolases (genetics)
• Puromycin (metabolism)