Regulation Of Sugar Transport And Metabolism In Oral Bac

饮食中的蔗糖是导致龋齿的主要因素。这种双糖(由葡萄糖和果糖分子组成)提供了合成多糖的前体，促进了链球菌在牙齿表面的黏附。乳酸的局部产生(通过蔗糖的微生物发酵)会导致表面牙釉质脱矿，并引发龋齿。改变葡萄糖部分的C_1和果糖环上剩余的五个羟基之间的O-糖苷键的位置，可以得到蔗糖的五个立体异构体：海藻糖、透壳糖、麦芽糖、白砂糖和棕榈糖。在我们的研究计划开始之前，人们通常认为微生物不能代谢蔗糖的异构体。这种信念，以及异构体相对较甜的事实，鼓励了这些不会致龋齿的药物的使用。在各种食品中作为蔗糖替代品的化合物。令人惊讶的是，与预期相反的是，我们发现来自革兰氏阳性和革兰氏阴性属的许多细菌物种(包括肺炎克雷伯菌、枯草芽孢杆菌、乙酰丁酸梭菌和Mortiferum梭杆菌)很容易在这五种异构体双糖上生长。有能力代谢蔗糖异构体的细菌总是有两个染色体基因编码：(I)依赖于膜的磷酸烯醇式丙酮酸(PEP)转运蛋白，促进异构体的同时转位和磷酸化；(Ii)独特的依赖NAD/Mn(2+)的磷酸-α-葡萄糖苷酶，催化磷酸化异构体在细胞内水解制得葡萄糖6-磷酸和果糖。在肺炎克雷伯菌的例子中，两个基因(ag1a，aglB)及其产物分别被命名为ag1a和ag1B。虽然肺炎克雷伯菌和其他物种是生长的先决条件，但目前还不清楚这两种蛋白单独表达是否足以在异构体双糖上生长微生物。尤其令人担忧的是，这些特定物种缺乏编码EIIA(AGL)的基因，EIIA是依赖于PEP的AGL：磷酸转移酶系统(PTS)功能活动所需的第三种可磷酸化蛋白质。以前(但没有证据)，我们建议来自不同PTS的EIIA必须取代丢失的？EIIA(AGL)，以补充PEP依赖的AGL转运系统的活性。为了解决这一假设，2006年与瑞士伯尔尼大学的研究人员共同发起了一项调查。在我们的研究中，选择了大肠杆菌K-12，因为这种肠道细菌不能代谢蔗糖或其异构体，很容易受到遗传操作的影响。重要的是，我们的合作者提供了包含必要突变的大肠杆菌K-12菌株以及磷酸转移酶系统的纯化组件(HPR和酶I)。我们的实验方法是首先将aglA和aglB基因克隆到表达载体(PAP2)中。当用该质粒转化时，大肠杆菌K-12很容易在多种O-α连接的葡萄糖苷上生长，包括蔗糖的所有异构体。随后，将pAP2转化到缺失EIIA(GLC)的葡萄糖：PTS突变株K-12中。该转化子不能在所测试的任何α-葡萄糖苷上生长，因此暗示EIIA(GLC)参与了蔗糖异构体的运输。用含有肺炎克雷伯菌agla转运蛋白的大肠杆菌K-12膜制剂进行的体外实验证实了我们的假设。当添加高能磷化供体(磷酸烯醇式丙酮酸)、HPR和EI时，这些细胞膜不能催化异构体的磷酸化。然而，当将纯化的EIIA(GLC)添加到反应混合物中时，观察到双糖立即和快速的磷酸化。我们认为葡萄糖-PTS的AGLA(转运体)、AglB(磷酸-α-葡萄糖苷酶)和EIIA(GLC)对于微生物在蔗糖异构体和相关的α-D-葡萄糖苷上的生长是必要和充分的。

Dietary sucrose is a major contributor to the etiology of dental caries. This disaccharide, (comprised of glucose and fructose molecules) provides the precursors for glycan synthesis, that facilitates adherence of Streptococci to the tooth surface. The localized production of lactic acid (via the microbial fermentation of sucrose) causes demineralization of surface enamel, and initiation of tooth decay. Positional change of the O-glycosidic linkage between C1 of the glucose moiety and the remaining five -OH groups of the fructose ring, yields the five stereo- isomers of sucrose: trehalulose, turanose, maltulose, leucrose and palatinose. Prior to commencement of our research program, it was generally assumed the microorganisms were unable to metabolize the isomers of sucrose. This belief, and the fact that the isomers are comparatively sweet, has encouraged the use of these ?non-cariogenic? compounds as substitutes for sucrose in various food products. Surprisingly, and contrary to expectation, we have found that many bacterial species from both Gram-positive and Gram-negative genera (including, Klebsiella pneumoniae, Bacillus subtilis, Clostridium acetobutylicum and Fusobacterium mortiferum) readily grow on these five isomeric disaccharides. Bacteria with the capacity to metabolize the isomers of sucrose invariably possess two chromosomal genes that encode: (i) a membrane-localized phosphoenolpyruvate (PEP)-dependent transporter that facilitates the simultaneous translocation and phosphorylation of the isomers, and (ii) a unique NAD/Mn(2+) -dependent phospho- alpha-glucosidase that catalyzes the intracellular hydrolysis of phosphorylated isomers to yield glucose 6-phosphate and fructose. In the case of Klebsiella pneumoniae, the two genes (aglA, aglB) and their products are designated AglA and AglB, respectively. Although prerequisite for growth of K. pneumoniae and other species, it was unclear whether expression of these two proteins alone, was sufficient for growth of microorganisms on the isomeric disaccharides. Of particular concern was the fact that these particular species lack a gene encoding EIIA(Agl), a third phosphorylatable protein required for functional activity of a PEP-dependent Agl: phosphotransferase system (PTS). Previously (but without proof), we suggested that an EIIA from a different PTS must substitute for the ?missing? EIIA(Agl) in order to complement activity of the PEP-dependent Agl transport system. To address this hypothesis, a collaborative investigation was initiated in 2006 with researchers at the University of Berne, Switzerland. For our studies, Escherichia coli K-12 was the organism of choice, because this enteric bacterium is unable to metabolize sucrose or its isomers, and is readily amenable to genetic manipulation. Importantly, strains of E. coli K-12 containing the necessary mutations, and purified components of the phosphotransferase system (HPr and Enzyme I) were made available by our collaborators. Our experimental approach was to first clone both aglA and aglB genes into an expression plasmid (pAP2). When transformed with this plasmid, E. coli K-12 grew readily on a wide variety of O-alpha-linked glucosides, including all isomers of sucrose. Subsequently, a mutant strain of E. coli K-12 lacking EIIA(Glc) of the glucose: PTS was also transformed with pAP2. This transformant failed to grow on any of the alpha-glucosides tested, thus suggestive of EIIA(Glc) participation in the transport of sucrose isomers. Confirmation for our hypothesis was obtained from in vitro experiments conducted with membrane preparations of the E. coli K-12 containing the AglA transporter from K. pneumoniae. When supplemented with the high-energy phosphoryl donor (phosphoenolpyruvate), HPr and EI, these cytoplasmic membranes failed to catalyze the phosphorylation of the isomeric compounds. However, upon addition of purified EIIA(Glc) to the reaction mixture, an immediate and rapid phosphorylation of the disaccharides was observed. We believe that AglA (transporter), AglB (phospho-alpha -glucosidase), and EIIA(Glc) of the glucose-PTS are necessary and sufficient, for the growth of microorganisms on sucrose isomers and related alpha-D-glucopyranosides.