INSERTIONAL INACTIVATION OF THE GENE ENCODING A 76-KILODALTON CELL-SURFACE POLYPEPTIDE IN STREPTOCOCCUS-GORDONII CHALLIS HAS A PLEIOTROPIC EFFECT ON CELL-SURFACE COMPOSITION AND PROPERTIES
INSERTIONAL INACTIVATION OF THE GENE ENCODING A 76-KILODALTON CELL-SURFACE POLYPEPTIDE IN STREPTOCOCCUS-GORDONII CHALLIS HAS A PLEIOTROPIC EFFECT ON CELL-SURFACE COMPOSITION AND PROPERTIES
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DOI：
10.1128/iai.58.11.3689-3697.1990
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发表时间：
1990-11-01
影响因子：
3.1
通讯作者：
EASINGWOOD, RA
中科院分区：
文献类型：
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作者：
JENKINSON, HF;EASINGWOOD, RA
在λgt11中构建S.gordonii DL1-Challis DNA文库。筛选噬菌斑以产生与戈氏链球菌细胞表面蛋白的抗血清反应的抗原。分离出称为λgt11-cp2的重组噬菌体，其携带1.85kb的戈登沙门氏菌DNA并在大肠杆菌中表达分子量为29kDa的抗原。与表达产物反应的抗体经过亲和纯化，并显示与 S. gordonii DL1-Challis 中分子量为 76 Da 的单一多肽抗原反应。将转录单元内的克隆DNA片段（0.85 kb）连接到携带红霉素抗性决定簇的非复制质粒中，并转化到S. gordonii DL1-Challis中。质粒整合到染色体上，并且76-kDa多肽抗原的表达被废除。基因失活对细菌生长或许多表型特性（包括疏水性和粘附性）没有明显影响。然而，它消除了血清诱导的细胞聚集，突变细胞降低了唾液和初乳免疫球蛋白 A 中的聚集滴度，并且还减少了与某些放线菌属物种的共聚集。来自野生型和突变株的细胞包膜蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱表明，除了缺乏表面暴露的 76-kDa 多肽外，突变型细胞包膜还缺乏几种其他多肽，包括与免疫球蛋白 A 结合的多肽。编码 76-kDa 多肽的基因在金黄色葡萄球菌中的表达。 gordonii 似乎对于细胞表面的功能构象至关重要。
A library of S. gordonii DL1-Challis DNA was constructed in .lambda.gt11. Phage plaques were screened for production of antigens that reacted with antiserum to S. gordonii cell surface proteins. A recombinant phage denoted .lambda.gt11-cp2 was isolated that carried 1.85 kb of S. gordonii DNA and that expressed an antigen with a molecular mass of 29 kDa in Escherichia coli. Antibodies that reacted with the expression product were affinity purified and were shown to react with a single polypeptide antigen with a molecular mass of 76 Da in S. gordonii DL1-Challis. A segment (0.85 kb) of the cloned DNA within the transcription unit was ligated into a nonreplicative plasmid carrying an erythromycin resistance determinant and transformed into S. gordonii DL1-Challis. The plasmid integrated onto the chromosome, and expression of the 76-kDa polypeptide antigen was abolished. The gene inactivation had no obvious effect on bacterial growth or on a number of phenotypic properties, including hydrophobicity and adherence. However, it abolished serum-induced cell aggregation, mutant cells had reduced aggregation titers in saliva and in colostrum immunoglobulin A, and it also reduced coaggregation with some Actinomyces species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of cell envelope protein from wild-type and mutant strains showed that as well as lacking the surface-exposed 76-kDa polypeptide, mutant cell envelopes were deficient in several other polypeptides, including those that bound to immunoglobulin A. Expression of the gene encoding the 76-kDa polypeptide in S. gordonii appeared to be critical for functional conformation of the cell surface.