Identification of Two 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 nature and the location of the secretion signal(s) in PulA presumed to be necessary for its specific secretion. The starting material was a gene fusion coding for an efficiently secreted PulA-beta-lactamase hybrid protein. Successive series of exonuclease III-generated deletions were used to remove internal segments of PulA from this hybrid. A simple plate test allowed the identification of truncated hybrids that retained beta-lactamase activity and that were secreted. Two non-adjacent regions, A and B (78 and 80 amino acids, respectively), were together necessary and sufficient to promote beta-lactamase translocation across the outer membrane. Secretion of PulA itself was markedly reduced when either of these regions was deleted, and was completely abolished when both regions were eliminated.