Heterologous expression and cellular distribution in Escherichia coli of the gene from Klebsiella pneumoniae which encodes alginate lyase.

The inducible alginate lyase of Klebsiella ptieitmoniue catalyses the degradation of guluronate-rich regions of the polysaccharide alginate [ I ] . The alginate lyase, which is chromosomally encoded [ 21, is largely secreted into the medium (31. although intracellular forms of the enzyme have been reported [4, 51. Recently, we have reported the cloning and heterologous expression in strains of Escherichia coli of the alginate lyase from K. ptieumorziae [3]. We now describe experiments to establish the cellular localization of the enzyme in the original strain of K. przeitmorziae and in recombinant strains of E. coli. A stable recombinant cosmid, pSP1, contained 12.1 kb of insert DNA from K. prieirmoriiue and resulted in the expression of alginate lyase activity in strains of E. coli [3] . The gene encoding the alginate lyase, aly, was located on a 1.05 kb !firidlll fragment, which was subcloned into an expression vector, pHG327 to produce the recombinant plasmid, pRCS 131. These constructs were used to study the expression of the recombinant alginate lyase in various strains of E. coli. Alginate lyase activity was quantified using the 3,sdinitrosalicylic acid reagent to measure the increase in reducing sugar end-groups [ 6 ] . One unit of activity is defined as the release of 1 pmol of reducing sugar/min under the conditions of assay [4]. Cytoplasmic, periplasmic and membrane fractions of the bacteria (obtained from 30 ml samples of the cultures) were prepared and analysed for their characteristic marker enzymes, /3-galactosidase, alkaline phosphatase and NADH oxidase, respectively, as described previously [ 71. The analysis of alginate lyase distribution at a single point during the growth of cells showed that approximately 70% of the enzyme was extracellular in late log phase cultures of K. pneumoniae. The majority of the remaining activity was localized in the cytosol. Strains of E. coli containing pSPl showed a similar distribution pattern to that of K . pneumotiiae. In contrast, the strain of E. coli containing the subcloned plasmid pRCS produced different results with approximately 70% of the alginate lyase being retained in the periplasm and 25% in the cytoplasm. The study was expanded to consider the cellular distribut ion of the alginate lyase during different stages of growth. The relative amounts of alginate lyase present in the extracellular fraction of K. pneumoniae cultures decreased progressively during growth (Fig. 1 a) . The greatest amount of lyase was produced during early stationary phase with the majority of the enzyme localized in the periplasm and the cytosol. With strains of E. coli containing pRCS, the enzyme was localized principally in the periplasm and cytosol (Fig. 1 h). However, during early log phase the greatest proportion of the small quantity of lyase that was synthesized was exported into the medium. The distribution of alginate lyase in K. pneumoniae, and the polymeric nature of the substrate, imply that the enzyme is actively exported by the bacteria. Maximal extracellular levels of the enzyme occur during late log phase rather than stationary phase, indicating that the alginate lyase is actively