SECRETION OF ACTIVE BETA-LACTAMASE TO THE MEDIUM MEDIATED BY THE ESCHERICHIA-COLI HEMOLYSIN TRANSPORT PATHWAY

An in frame gene fusion containing the coding region for mature beta-l actamase and the 3'-end of hylA encoding the haemolysin secretion sign al, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external mediu m in the presence of the haemolysin translocator proteins, HlyB and Hl yD. The specific activity of the beta-lactamase portion of the secrete d protein (measured by the hydrolysis of penicillin G), approximately 1 U/mu g protein, was close to that of authentic, purified TEM-beta-la ctamase. This is an important example of a hybrid protein that is enzy matically active, and secreted via the haemolysin pathway. Previous st udies have indicated that haemolysin is secreted directly into the med ium, bypassing the periplasm, to which beta-lactamase is normally targ eted. This study indicated, therefore, that normal folding of an activ e beta-lactamase, can occur, at least when fused to the HlyA C-terminu s, without the necessity of entering the periplasm. Despite the secret ion of approximately 5 mu g/ml. levels of the active beta-lactamase fu sion into the medium, there was maximally only a 50% detectable increa se in the LD(50) for resistance to ampicillin at the individual cell l evel. This result suggests that, normally, resistance to ampicillin re quires a high concentration of the enzyme close to killing targets, i. e. in the periplasm, in order to achieve significant levels of protect ion.