Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis
Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis
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DOI：
10.1128/iai.68.5.2475-2483.2000
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发表时间：
2000-05-01
影响因子：
3.1
通讯作者：
Hamada, S
中科院分区：
文献类型：
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作者：
Fujiwara, T;Hoshino, T;Hamada, S
口腔链球菌是口腔链球菌家族的成员，是人类口腔中的早期定植微生物。S.已知口腔链球菌产生葡糖基转移酶（GT酶），其从蔗糖合成葡聚糖。该酶通过色谱法从口腔链球菌ATCC 10557的培养物上清液中纯化。纯化的酶，GTase-R，具有173 kDa的分子量和6.3的pI。该酶主要合成水溶性葡聚糖，没有引物依赖性。GTase的加入显著增强了变形链球菌的蔗糖依赖性静息细胞粘附，其水平与生长中的变形链球菌细胞相似。变异体抗GTase-R抗体对戈登链球菌和血链球菌的葡聚糖合成活性有抑制作用，对S.口腔GTase-R的N-末端氨基酸序列与已知的口腔链球菌GTase序列没有相似性。利用简并PCR引物，克隆了一个8.1kb的DNA片段，该片段携带编码GTase-R的基因（gtfR）及其调节基因（rgg）。推导的氨基酸序列比较表明，S. oralis和S. gordonii的亲缘关系较近。发现gtfR基因具有对应于N末端130个氨基酸残基的物种特异性核苷酸序列。在rgg或gtfR基因中插入aphA或aph A导致生物体的GTase活性降低，并改变了这些转化体的殖民地形态。这些结果表明S.口腔GT酶可能在变形链球菌随后的定殖中起重要作用。
Streptococcus oralis is a member of the oral streptococcal family and an early-colonizing microorganism in the oral cavity of humans. S. oralis is known to produce glucosyltransferase (GTase), which synthesizes glucans from sucrose, The enzyme was purified chromatographically from a culture supernatant of S, oralis ATCC 10557. The purified enzyme, GTase-R, had a molecular mass of 173 kDa and a pI of 6.3. This enzyme mainly synthesized water-soluble glucans with no primer dependency. The addition of GTase markedly enhanced the sucrose-dependent resting cell adhesion of Streptococcus mutans at a level similar to that found in growing cells of S. mutans. The antibody against GTase-R inhibited the glucan-synthesizing activities of Streptococcus gordonii and Streptococcus sanguis, as well as S. oralis. The N-terminal amino acid sequence of GTase-R exhibited no similarities to known GTase sequences of oral streptococci. Using degenerate PCR primers, an 8.1-kb DNA fragment, carrying the gene (gtfR) coding for GTase-R and its regulator gene (rgg), was cloned and sequenced. Comparison of the deduced amino acid sequence revealed that the rgg genes of S. oralis and S, gordonii exhibited a close similarity. The gtfR gene was found to possess a species-specific nucleotide sequence corresponding to the N-terminal 130 amino acid residues. Insertion of erm or aphA into the rgg or gtfR gene resulted in decreased GTase activity by the organism and changed the colony morphology of these transformants. These results indicate that S. oralis GTase may play an important role in the subsequent colonizing of mutans streptoccoci.