Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli
| Autor: | J. C. Lazzaroni, Mark A. Blight, A. L. Pimenta, L. Kotelevets, S. J. Séror, C. Dando, I. B. Holland |
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| Rok vydání: | 1994 |
| Předmět: |
Proline
Recombinant Fusion Proteins Molecular Sequence Data Mutant Glycine Biology medicine.disease_cause Hemolysin Proteins Viral Proteins Bacterial Proteins Leucine Vancomycin Escherichia coli Genetics medicine Point Mutation Viral Regulatory and Accessory Proteins Secretion Amino Acid Sequence Molecular Biology Adenosine Triphosphatases chemistry.chemical_classification Aspartic Acid Mutation Base Sequence Sequence Homology Amino Acid Escherichia coli Proteins Mutagenesis Temperature Membrane Proteins Drug Resistance Microbial Periplasmic space Molecular biology Amino acid DNA-Binding Proteins Repressor Proteins Directed mutagenesis chemistry Biochemistry Genes Bacterial Mutagenesis Site-Directed ATP-Binding Cassette Transporters Carrier Proteins Sequence Alignment Signal Transduction |
| Zdroj: | Molecular and General Genetics MGG. 245:431-440 |
| ISSN: | 1432-1874 0026-8925 |
| Popis: | We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Gly10Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the lambda Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process. |
| Databáze: | OpenAIRE |
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