IDENTIFICATION OF 2 REGIONS OF KLEBSIELLA-OXYTOCA PULLULANASE THAT TOGETHER ARE CAPABLE OF PROMOTING BETA-LACTAMASE SECRETION BY THE GENERAL SECRETORY PATHWAY

Pullulanase (PulA) is a 116 kDa amylolytic lipoprotein secreted by the Gram-negative bacterium Klebsiella oxytoca via the general secretory pathway. A deletion strategy was used in an attempt to determine the n ature and the location of the secretion signal(s) in PulA presumed to be necessary for its specific secretion. The starting material was a g ene fusion coding for an efficiently secreted PulA-beta-lactamase hybr id protein. Successive series of exonuclease III-generated deletions w ere used to remove internal segments of PulA from this hybrid. A simpl e plate test allowed the identification of truncated hybrids that reta ined beta-lactamase activity and that were secreted. Two nonadjacent r egions, A and B (78 and 80 amino acids, respectively), were together n ecessary and sufficient to promote beta-lactamase translocation across the outer membrane. Secretion of PulA itself was markedly reduced whe n either of these regions was deleted, and was completely abolished wh en both regions were eliminated.