BETA-GALACTOSIDASE IS INACTIVATED BY INTERMOLECULAR DISULFIDE BONDS AND IS TOXIC WHEN SECRETED TO THE PERIPLASM OF ESCHERICHIA-COLI

The wild-type LamB-LacZ hybrid protein inhibits the export machinery u pon induction when assayed by biochemical and genetic techniques, a ph enotype referred to as hybrid protein jamming. This hybrid protein als o renders cells sensitive to growth in the presence of the inducer mal tose, presumably because of the jamming. We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein. This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C. Extreme C- terminal sequences of LacZ function as a critical folding domain and a re therefore responsible for stabilizing the LacZ structure. To determ ine if this region of LacZ is important for jamming, we recombined a l ate nonsense mutation (X90) onto the hybrid construct. We found the to xicity of this new hybrid, LamB-LacZX90, to be nearly identical to tha t of the full-length protein, but it also does not jam the secretion m achinery. This suggests that jamming is caused by LacZ folding. We fou nd no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is n ovel. Under these conditions, the tripartite and X90 fusion proteins f orm disulfide-bonded aggregates with high molecular weights in the per iplasm. Accordingly, we believe that LacZ disrupts some essential func tion(s) in the periplasm.