IDENTIFICATION AND PRELIMINARY CHARACTERIZATION OF TEMPERATURE-SENSITIVE MUTATIONS AFFECTING HLYB, THE TRANSLOCATOR REQUIRED FOR THE SECRETION OF HEMOLYSIN (HLYA) FROM ESCHERICHIA-COLI

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesi s. 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 substitu tion, a derivative of HlyB, in which the first 25 amino acids were rep laced by 21 amino acids of the lambda Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispens able for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a condi tional lethal mutation in HlyB. We have isolated 4 additional mutation s in PIV by directed mutagenesis, giving a total of 5 out of 12 residu es substituted in this region, with 4 mutations rendering HlyB defecti ve in secretion. The Pro624 mutation, close to the Walker B-site for A TP binding in the cytoplasmic domain is identical to a mutation in His P that leads to uncoupling of ATP hydrolysis from the transport of his tidine. 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 a lone. Thus, active translocation of HlyA renders the cells hyperpermea ble to the drug. Mutations in hlyB affecting secretion could be assign ed to two classes: those that restore the level of vancomycin resistan ce to that of E. coli not secreting HlyA and those that still confer h ypersensitivity to the drug in the presence of HlyA. We propose that m utations that promote vancomycin resistance will include mutations aff ecting initial recognition of the secretion signal and therefore activ ation of a functional transport channel. Mutations that do not alter H lyA-dependent vancomycin sensitivity may, in contrast, affect later st eps in the transport process.