This study has shown that strain K21 differs from the previously characterized and closely related strain PAP996 in that expression of the pullulanase gene () and other genes of the maltose regulon is partially independent of exogenous inducer (maltose/maltotriose). Mutants of strain K21 which are defective in pullulanase synthesis and/or secretion were isolated following Tn mutagenesis. Three phenotypic classes of mutants were identified. Class I mutants were defective in the surface localization and secretion of pullulanase. Class II mutants did not secrete detectable levels of pullulanase but were able to export pullulanase to the cell surface. Class II mutants also expressed pullulanase and other maltose-regulated genes at markedly lower levels than those found in the parent strain under non-inducing conditions. The single class III mutant was intermediate between K21 and class I mutants; most of the cell-associated pullulanase was localized at the cell surface whilst a significant amount was secreted into the medium. Mapping indicated that all but three of the Tn insertions were adjacent to, and at either side of, . One class II mutant carried a Tn insertion in or close to whereas in the remaining class II mutants the insertions were located at least 4 kb upstream of in a region which may define a new regulatory locus of the maltose operon.


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