d'Enfert C, Chapon C, Pugsley A P
Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
Mol Microbiol. 1987 Jul;1(1):107-16. doi: 10.1111/j.1365-2958.1987.tb00534.x.
Pullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze-thaw disruption of these cells released both membrane-associated and apparently soluble forms of pullulanase. Membrane-associated pullulanase co-fractionated with authentic outer membrane vesicles upon isopycnic sucrose-gradient centrifugation, whereas the quasi-soluble form had the same equilibrium density as inner membrane vesicles and extracellular pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the pullulanase structural gene (pulA) produced increased amounts of cell-associated and secreted pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for pullulanase secretion were saturated by the over-produced pullulanase. When pulA was expressed under lacZ promotor control, the pullulanase which was produced was not exposed on the cell surface at any time, suggesting that pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like pulA, may be part of the maltose regulon.
支链淀粉酶是肺炎克雷伯菌分泌的一种脂蛋白，蛋白酶、底物可及性和免疫荧光试验表明，它最初定位于外膜的外表面。这些细胞经冻融裂解后，释放出与膜相关的支链淀粉酶以及明显呈可溶形式的支链淀粉酶。在等密度蔗糖梯度离心中，与膜相关的支链淀粉酶与真正的外膜囊泡共分离，而准可溶形式的支链淀粉酶与内膜囊泡和细胞外支链淀粉酶聚集体具有相同的平衡密度。后者还含有外膜麦芽糖孔蛋白，但基本上不含包括脂多糖和脂质在内的其他膜成分。携带多个支链淀粉酶结构基因（pulA）拷贝的肺炎克雷伯菌产生的细胞相关和分泌型支链淀粉酶量增加，但即使长时间孵育后，很大一部分酶既未暴露在细胞表面，也未释放到培养基中。这表明过量产生的支链淀粉酶使支链淀粉酶分泌所需的因子饱和。当pulA在lacZ启动子控制下表达时，所产生的支链淀粉酶在任何时候都未暴露在细胞表面，这表明支链淀粉酶分泌基因不是组成型表达的，并且增加了它们可能像pulA一样是麦芽糖调节子一部分的可能性。
