INSERTIONAL INACTIVATION OF THE STREPTOCOCCUS-MUTANS DEXA (DEXTRANASE) GENE RESULTS IN ALTERED ADHERENCE AND DEXTRAN CATABOLISM
INSERTIONAL INACTIVATION OF THE STREPTOCOCCUS-MUTANS DEXA (DEXTRANASE) GENE RESULTS IN ALTERED ADHERENCE AND DEXTRAN CATABOLISM
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DOI：
10.1099/13500872-141-11-2929
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发表时间：
1995-11-01
期刊：
影响因子：
2.8
通讯作者：
RUSSELL, RRB
中科院分区：
文献类型：
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作者：
COLBY, SM;WHITING, GC;RUSSELL, RRB
变形链球菌能够从蔗糖合成细胞外葡聚糖，这有助于这些细菌的粘附。胞外葡聚糖酶可以部分降解葡聚糖，因此可能影响变形链球菌的毒力。为了分离不能产生葡聚糖酶的突变体，我们将随机从变形链球菌中提取的sau3al酶切的染色体DNA片段插入链球菌整合载体pVA891的BamHI位点，构建了一个DNA文库，该载体能够在大肠杆菌中复制，但不具有链球菌起源的复制。将合成的质粒导入S. mutans LT11中，通过同源重组实现插入失活。鉴定出两个不具有葡聚糖酶活性的转化子。质粒的一个拷贝整合到这些转化体的染色体上被南方杂交分析证实。利用标记拯救技术恢复质粒两侧的染色体DNA片段，并对其进行测序。用BLASTX程序与已知序列进行比较，结果显示测序后的基因片段在氨基酸水平上与sobrinus链球菌的葡聚糖酶有56%的同源性，强烈提示S. mutans葡聚糖酶基因（dexA）已失活。当在含有蔗糖的托德-休伊特琼脂上生长时，与亲本菌株相比，葡聚糖酶突变体的菌落形态发生了变化，细胞外聚合物明显增加。突变体也更倾向于平稳的海浪；但在蔗糖依赖性细胞-细胞聚集方面无明显差异。与LT11相反，dexA突变体和编码葡聚糖葡萄糖苷酶的dexB基因突变体都不能使葡聚糖发酵产生酸，这支持了先前的假设，即两种酶都是葡聚糖代谢所必需的。通过灭活葡聚糖基因得到的结果表明，葡聚糖酶在控制胞外葡聚糖的数量和性质、变形链球菌的粘附以及葡聚糖作为碳水化合物来源的利用方面发挥着作用。
Streptococcus mutans is able to synthesize extracellular glucans from sucrose which contribute to adherence of these bacteria. Extracellular dextranase can partially degrade the glucans, and may therefore affect virulence of S. mutans. In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random Sau3Al-digested fragments of chromosomal DNA from S. mutans into the BamHI site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia coli but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into S. mutans LT11, allowing insertional inactivation through homologous recombination. Two transformants were identified which did not possess dextranase activity. Integration of a single copy of the plasmid into the chromosome of these transformants was confirmed by Southern hybridization analysis. Chromosomal DNA fragments flanking the plasmid were recovered using a marker rescue technique, and sequenced. Comparison with known sequences using the BLASTX program showed 56% homology at the amino acid level between the sequenced gene fragment and dextranase from Streptococcus sobrinus, strongly suggesting that the S. mutans dextranase gene (dexA) had been inactivated. The colony morphology of the dextranase mutants when grown on Todd-Hewitt agar containing sucrose was altered compared to the parent strain, with an apparent build-up of extracellular polymer. The mutants were also more adherent to a smooth surf;ace than LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation. In contrast to LT11, neither the dexA mutants nor a mutant in the dexB gene, which encodes a dextran glucosidase, were able to ferment dextran to produce acid, supporting an earlier hypothesis that both enzymes are required for metabolism of dextran. From the results obtained by inactivating the dexA gene, a role for dextranase is suggested in controlling the amount and nature of extracellular glucans, in adherence of S. mutans, and in the utilization of glucans as a carbohydrate source.