ISOLATION AND CHARACTERIZATION OF THE STREPTOCOCCUS-MUTANS GTFD GENE, CODING FOR PRIMER-DEPENDENT SOLUBLE GLUCAN SYNTHESIS
ISOLATION AND CHARACTERIZATION OF THE STREPTOCOCCUS-MUTANS GTFD GENE, CODING FOR PRIMER-DEPENDENT SOLUBLE GLUCAN SYNTHESIS
复制标题
DOI：
10.1128/iai.57.7.2079-2085.1989
复制
发表时间：
1989-07-01
影响因子：
3.1
通讯作者：
KURAMITSU, HK
中科院分区：
文献类型：
--
作者：
HANADA, N;KURAMITSU, HK
变形链球菌GS-5的两个葡萄糖转移酶基因gtfb和gtfc已在本实验室分离并测序。在本通讯中，第三个GTF基因gtfD被分离和鉴定。该基因的分离涉及到一种利用整合质粒pVA891的新程序。用抗葡萄糖基转移酶抗体在pUC18克隆文库中初步鉴定了NHS1株1.7kb碱基DNA片段(含有gtfb和gtfc基因缺失)所表达的多肽。将该片段与pVA891连接后整合到GS-5染色体上，转化获得菌株DP2。经EcoRI酶切、鉴定和转化后，将gtfD基因的1个完整拷贝和1个不完整拷贝从菌株DP2的染色体上移除。HB101。构建的表达载体pNH4表达葡萄糖转移酶S(GTF-S)活性。该酶被纯化到接近均一的水平，并被证明以一种依赖于引物的方式专门合成水溶性葡聚糖。纯化的酶的相对分子质量(155kodalton)和动力学参数与先前从菌株GS-5的培养液中纯化的GTf-S酶相似。插入失活的gftD基因表明，该基因不是依赖蔗糖的体外黏附光滑表面所必需的。此外，GFTC GTFB突变体中gftD基因的失活表明，在变形链球菌GS-5染色体上存在三个与葡聚糖形成有关的不同的GTF基因。Southern杂交分析进一步表明，gtfD基因与血链球菌和远缘链球菌的gft基因没有明显的同源性。
Two glucosyltransferase genes from Streptococcus mutans GS-5, gtfB and gtfC, have been previously isolated and sequenced in this laboratory. In the present communication a third gtf gene, gtfD, was isolated and characterized. Isolation of the gene involved a novel procedure utilizing the integration plasmid pVA891. A peptide expressed by the 1.7-kilobase DNA fragment from strain NHS1 (containing deletions in both the gtfB and gtfC genes) was initially identified in a pUC18 clone bank with antiglucosyltransferase antibodies. This fragment was integrated into the GS-5 chromosome following ligation into pVA891 and transformation, yielding strain DP2. The vector together with one complete and one incomplete copy of the gtfD gene was removed from the chromosome of strain DP2 following EcoRI digestion, religation, and transformation of E. coli. HB101. The resultant plasmid, pNH4, expressed glucosyltransferase S (GTF-S) activity. The enzyme was purified to near homogeneity and was shown to synthesize water-soluble glucan exclusively in a primer-dependent manner. The molecular mass (155 kilodaltons) and the kinetic parameters of the purified enzyme were similar to those observed for the GTF-S enzyme previously purified from culture fluids of strain GS-5. Insertional inactivation of the gftD gene indicated that this gene is not required for in vitro sucrose-dependent adherence to smooth surfaces. Furthermore, inactivation of the gftD gene in a gftC gtfB mutant indicated that three distinct gtf gene involved in glucan formation are present on the S. mutans GS-5 chromosome. Southern blot analysis further suggested that the gtfD gene does not share demonstrable homology with the gft genes from Streptococcus sanguis or Streptococcus sobrinus.