Castuma C E, Huang R, Kornberg A, Reusch R N
Department of Biochemistry, Stanford University School of Medicine, California 94305, USA.
J Biol Chem. 1995 Jun 2;270(22):12980-3. doi: 10.1074/jbc.270.22.12980.
A complex of polyhydroxybutyrate (PHB), Ca2+, and inorganic polyphosphate (polyP) was proposed as the membrane component responsible for competence for DNA entry in Escherichia coli (Reusch, R. N., and Sadoff, H. L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4176-4180). While chemical and immunological assays and 1H NMR have unequivocally established the identity and content of PHB in the complex, comparable methods were not available for polyP. With specific enzyme assays developed for polyP, we have identified, in chloroform extracts of competent cell membranes, a novel form of polyP of about 60 to 70 residues in a stoichiometric ratio of PHB to polyP of 2:1. In E. coli mutants, incapable of synthesizing the predominant, thousand-long polyP chains, appearance of this short polyP and its inclusion in membranes can account for their capacity to develop competence and indicates an auxiliary pathway for polyP synthesis. A variety of fluorescent lipid probes demonstrate the appearance of extensive rigid domains in membranes of competent cells. We propose that the PHB.Ca2+.polyP complex perturbs the conformation of the lipid matrix, making it more permeable to charged molecules and thus allowing the entry of DNA.
聚羟基丁酸酯（PHB）、Ca2+和无机多聚磷酸盐（polyP）的复合物被认为是大肠杆菌中负责DNA进入能力的膜成分（Reusch，R. N.，和Sadoff，H. L.（1988年）《美国国家科学院院刊》85，4176 - 4180）。虽然化学和免疫分析以及1H NMR已经明确确定了复合物中PHB的身份和含量，但对于polyP却没有类似的可用方法。通过为polyP开发的特定酶分析方法，我们在感受态细胞膜的氯仿提取物中鉴定出一种新型的polyP，其长度约为60至70个残基，PHB与polyP的化学计量比为2:1。在无法合成主要的、长达数千个残基的polyP链的大肠杆菌突变体中，这种短polyP的出现及其包含在膜中可以解释它们产生感受态的能力，并表明存在polyP合成的辅助途径。多种荧光脂质探针显示感受态细胞膜中出现了广泛的刚性区域。我们提出，PHB.Ca2+.polyP复合物扰乱了脂质基质的构象，使其对带电分子更具渗透性，从而允许DNA进入。
