Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4
Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4
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DOI：
10.1128/aem.62.1.196-202.1996
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发表时间：
1996-01-01
影响因子：
4.4
通讯作者：
Shoemaker, NB
中科院分区：
文献类型：
--
作者：
Gardner, RG;Russell, JB;Shoemaker, NB
通过添加fusca热孢子菌纤维素酶的纤维素结合结构域，重建了瘤胃普雷votella B(1)4的羧甲基纤维素酶（CMCase）基因，并利用氯霉素和四环素抗性穿梭载体（pTC-COW）从大肠杆菌偶联转移到均匀拟杆菌0061上。pTC-COW是专门构建的载体，用于从均匀拟杆菌供体偶联转移到瘤胃拟杆菌受体。含有CMCase结构的均匀杆菌转偶联物克隆到表达Cm-r的pTC-COW中，但直到在CMCase上游加入P. ruminicola 23的木聚糖酶启动子（pTC-XRCMC）后，它们才产生重构的CMCase。木聚糖酶启动子允许均匀杆菌转偶联物产生大量重构的CMCase，并出现在细胞的外表面。虽然重建的CMCase单独不允许均匀芽孢杆菌在酸胀的纤维素上生长，但当在培养上清中加入两种胞外酶时，可以观察到快速生长。在这些条件下，重建的CMCase比野生型CMCase生长得更快。在严格的厌氧条件下，采用硝化纤维素过滤器（细胞固定化）和更严格的选择，pTC-XRCMC从均匀B转移到P，瘤胃B(1)4的频率提高了100倍。虽然P. ruminicola B(1)4 （pTC-XRCMC）转偶联物表达Tc-r，并具有与穿梭载体探针杂交的DNA，但即使在木聚糖为碳源时，这些转偶联物也不能产生可检测水平的重建CMCase。基于这些结果，似乎并不是所有被B、uniform和P. ruminicola 23识别的启动子都在P. ruminicola B(1)4中起作用，然而，B. uniform的结果表明，P. ruminicola B(1)4启动子的引入应该允许重组的CMCase在P. ruminicola B(1)4中表达。
A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1)4 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW), pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients, B, uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cm-r, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC), The xylanase promoter allowed the B. uniformis transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells, Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid-swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. tinder these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P, ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocellulose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B(1)4 (pTC-XRCMC) transconjugants expressed Tc-r and had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was the carbon source. On the basis of these results, it appears that hot all of the promoters recognized by B, uniformis and P. ruminicola 23 are functional in P. ruminicola B(1)4, However, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstructed CMCase in P. ruminicola B(1)4.