Regulation Of Sugar Transport And Metabolism In Oral Bacteria

口服细毛菌发酵蔗糖异构体的研究 测序结果表明，口腔乳杆菌ATCC 14201基因组在Lebu_1525、1526和1527位点有3个相邻基因。这些基因的翻译产物分别与磷酸葡萄糖苷酶(PAGL)、调节蛋白(GntR)和磷酸烯醇丙酮酸依赖的糖运输蛋白(EIICB)有显著的同源性。在非口腔细菌物种中，这些基因包括促进蔗糖异构体代谢的sim操纵子。生长研究表明，口腔乳杆菌能发酵多种碳水化合物，包括蔗糖的五种异构体中的四种。在异构体双糖上的生长诱导了一个大小与Lebu_1525编码的多肽相当的50 kDa多肽的表达。克隆了后者的基因，并从大肠杆菌TOP 10细胞中纯化了表达的蛋白。在两个基本辅因子(NAD+和Mn2+)的存在下，该酶很容易水解对硝基苯基-α-吡喃葡萄糖苷6-磷酸(PNPalphaG6P)，这是蔗糖的磷酸化异构体的显色类似物。通过比较序列比对、免疫反应性和特征基序，将该酶归属于糖基水解酶家族4的磷酸-葡萄糖苷酶(PAGL)。我们认为Lebu_1527和1525的产物分别催化蔗糖异构体的磷酸化易位和水解性。Leptotricia的四个遗传多样性，但16S rDNA相关的种最近被描述：L.Good Fellowii，L.hofstadii，L.shahii和L.Wadei。我们对这些新物种在碳水化合物利用方面的表型特征进行了测定，得出了一些意想不到的发现。虽然非口腔细菌物种总是生长在所有蔗糖异构体上，但支持细毛菌特定物种生长的异构体数量(S)有相当大的差异。例如，沙氏乳杆菌能代谢所有5种异构体，口腔乳杆菌能发酵4种蔗糖，而瓦台乳杆菌在相同条件下不能在麦芽糖或白糖上生长。在分子水平上，这种异构体区分可能反映了Pag1蛋白运输或细胞内的微妙的物种特异性突变或构象变化。当细毛菌不同物种的基因组完成完整测序后，未来的比较分析可能会揭示代谢特征可变性的遗传基础。随着人类口腔微生物组数据库(HOMD)中遗传信息的积累，很可能在目前尚未测序的口腔细菌基因组中发现与细毛菌SIM操纵子类似的遗传位点。在这种情况下，由口腔微生物发酵推定的非致龋性双糖可能比目前设想的更广泛。我们的研究结果发表在同行评议期刊《分子口腔微生物学》上。 肺炎链球菌TIGR4磷酸β-葡萄糖苷酶(BGLA-2)的结构与功能 肺炎链球菌是急性肺炎、中耳炎、脑膜炎和败血症的主要病原体，每年导致全球数百万人死亡。在人类宿主中，肺炎链球菌会遇到各种糖偶联物，包括粘蛋白、宿主防御分子和上皮细胞表面暴露的多糖。与其他病原微生物一样，肺炎链球菌产生多种分泌性或表面相关的糖苷酶，其功能包括修饰和水解宿主糖结合物(S)。基因组测序，结合对新毒力因子的探索，表明大量的糖苷酶是肺炎链球菌最大毒力所必需的。BglA-2由肺炎链球菌TIGR4染色体DNA中的Sp_0578基因编码。将该基因克隆到高效表达载体BglA-2(471个残基，Mr=54,361)中，纯化后的蛋白为均一的电泳带。BglA-2的天然底物包括：纤维二糖-6-磷酸、龙胆二糖-6P、熊果苷-6P、水杨素-6P和相关的O-β连接的双糖磷酸酯。这些化合物不能在商业上获得，因此是通过我们实验室以前开发的程序酶法制备的(J.Biol。化学。2002年：34310-34321)。这些新化合物的使用使得底物专一性和动力学分析得以进行。用等温滴定量热法测定了BglA-2与底物结合的热变、配体亲和力和化学计量比等热力学参数。制备了BglA-2的天然晶体和与不可水解性类似物(硫代纤维二糖-6-磷酸)形成的络合物，并用X射线(同步加速器)衍射和分子置换拆分确定了该酶的结构。未来的实验，包括定点突变和基因缺失，将解决BglA-2对肺炎链球菌感染的致病作用(S)。

FERMENTATION OF SUCROSE ISOMERS BY ORAL LEPTOTRICHIA The sequenced genome of L. buccalis ATCC 14201 revealed three contiguous genes at loci: Lebu_1525,1526 and 1527. The translation products of these genes exhibited significant homology with phospho-α-glucosidase (Pagl), a regulatory protein (GntR) and a phosphoenol pyruvate-dependent sugar transport protein (EIICB), respectively. In non-oral bacterial species, these genes comprise the sim operon that facilitates sucrose isomer metabolism. Growth studies showed that L. buccalis fermented a wide variety of carbohydrates, including four of the five isomers of sucrose. Growth on the isomeric disaccharides elicited expression of a 50kDa polypeptide comparable in size to that encoded by Lebu_1525. The latter gene was cloned, and the expressed protein was purified to homogeneity from Escherichia coli TOP 10 cells. In the presence of two essential cofactors (NAD+ and Mn2+ ion) the enzyme readily hydrolyzed p-nitrophenyl-alpha-glucopyranoside 6-phosphate (pNPalphaG6P), a chromogenic analog of the phosphorylated isomers of sucrose. By comparative sequence alignment, immuno- reactivity and signature motifs, the enzyme was assigned to the phospho-α-glucosidase (Pagl) clade of glycosylhydrolase Family 4. It is our contention that the products of Lebu_1527 and 1525, catalyze the phosphorylative translocation and hydrolysis of sucrose isomers in L. buccalis, respectively. Four genetically diverse, but 16S rDNA related species of Leptotrichia have recently been described: L. goodfellowii, L. hofstadii, L. shahii and L. wadei. Our determination of the phenotypic traits of these new species with respect to carbohydrate utilization yielded some unexpected findings. Whereas non-oral bacterial species invariably grow on all sucrose isomers, considerable variation was noted in the number(s) of isomers that supported growth of a particular species of Leptotrichia. For example, L. shahii metabolized all five isomeric compounds, L. buccalis fermented four of the sucro-disaccharides whereas, under the same conditions, L.wadei failed to grow on either maltulose or leucrose. At the molecular level, such isomeric discrimination presumably reflects subtle species-specific mutations or conformational changes in either the transport or intracellular Pagl proteins. The genetic basis for the variability of metabolic traits may be revealed by future comparative analyses when the genomes of the different species of Leptotrichia have been sequenced in their entirety. As genetic information accumulates in the Human Oral Microbiome Database (HOMD), it likely that genetic loci similar to the sim operon in Leptotrichia will be discovered in presently un-sequenced genomes of oral bacteria. In this context, the fermentation of presumed non-cariogenic disaccharides by oral microorganisms may be more widespread than currently envisaged. Our findings have been published in the peer-reviewed journal, Molecular Oral Microbiology. STRUCTURE AND FUNCTION OF PHOSPHO-BETA-GLUCOSIDASE (BGLA-2) FROM STREPTOCOCCUS PNEUMONIAE TIGR4. Streptococcus pneumonia is the major causative agent of acute pneumonia, otitis media, meningitis, and septicemia, which annually result in the deaths of millions worldwide. In the human host, S. pneumoniae encounters a variety of glyco-conjugates, including mucins, host defense molecules, and surface exposed glycans on epithelial cells. In common with other pathogenic microbes, S. pneumonia produces a variety of secreted or surface-associated glycosidases whose function(s) include the modification and hydrolysis of host glyco-conjugates. Genome sequencing, in combination with exploration of new virulence factors, suggests that a large number of glycosidases are necessary for maximum virulence of S. pneumoniae. BglA-2 is encoded by the gene Sp_0578 in the chromosomal DNA of S. pneumoniae TIGR4. After cloning of the gene in a high expression vector, BglA-2 (471 residues, Mr = 54,361) was purified to electrophoretic homogeneity. The natural substrates of BglA-2 include: cellobiose-6-phosphate, gentiobiose-6P, arbutin-6P, salicin-6P and related O-beta-linked disaccharide phosphates. These compounds are not commercially available, and accordingly were prepared enzymatically by procedures developed previously in our laboratory (J. Biol. Chem. 277: 34310-34321, 2002). Use of these novel compounds permitted substrate specificity and kinetic analyses to be conducted. Thermodynamic parameters including enthalpy changes, ligand affinity and stoichiometry of binding between substrates and BglA-2 were determined by isothermal titration calorimetry (ITC). Crystals of BglA-2 in native form, and in complex with a non-hydrolyzable analog (thio-cellobiose-6-phosphate), have been prepared, and the structure of the enzyme has been solved by X-ray (synchrotron) diffraction and resolution of phase by molecular replacement. Future experiments, involving site-directed mutagenesis and gene deletion, will address the pathogenic contribution(s) of BglA-2 to S. pneumoniae infection.