Development and Application of a PCR-Targeted Gene Disruption Method for Studying CelR Function in Thermobifida fusca
Development and Application of a PCR-Targeted Gene Disruption Method for Studying CelR Function in Thermobifida fusca
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DOI：
10.1128/aem.02626-09
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发表时间：
2010-04-01
影响因子：
4.4
通讯作者：
Fong, Stephen S.
中科院分区：
文献类型：
--
作者：
Deng, Yu;Fong, Stephen S.
Thermobifida fusca是一种高G + C含量、嗜热、革兰氏阳性的土壤放线菌，具有较高的纤维素分解活性。于T.认为CelR通过与许多已知纤维素酶基因上游的14-bp反向重复序列[5 ′-（T）GGGAGCGCTCCC（A）]结合而作为纤维素酶基因表达的主要调节因子。以前，研究T. fusca在很大程度上受到缺乏针对T.褐色。在这项研究中，我们开发了一种有效的程序，用于创建精确的染色体基因破坏，并证明了这一过程中产生的celR缺失菌株。然后使用生长行为、纤维素酶活性和基因表达的测量来表征celR缺失菌株。T. Fusca表现出严重受损的生长表型，具有延长的滞后期，并且在葡萄糖和纤维二糖上生长的细胞产率降低。虽然最大内切葡聚糖酶活性和纤维素酶活性没有显着变化，但每个细胞的内切葡聚糖酶活性和纤维素酶活性大幅提高。在celR缺失菌株和野生型菌株中的mRNA转录水平的测量表明，CelR蛋白潜在地充当某些基因的阻遏物和其它基因的激活物。总之，我们建立并证明了一种在T. fusca，可用于研究该生物体的纤维素分解能力。这种方法的组成部分可能是有用的，在开发其他目前棘手的生物体的基因工程方法。
Thermobifida fusca is a high-G+C-content, thermophilic, Gram-positive soil actinobacterium with high cellulolytic activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat [5'-(T)GGGAGCGCTCCC(A)] that is upstream of many known cellulase genes. Previously, the ability to study the roles and regulation of cellulase genes in T. fusca has been limited largely by a lack of established genetic engineering methods for T. fusca. In this study, we developed an efficient procedure for creating precise chromosomal gene disruptions and demonstrated this procedure by generating a celR deletion strain. The celR deletion strain was then characterized using measurements for growth behavior, cellulase activity, and gene expression. The celR deletion strain of T. fusca exhibited a severely crippled growth phenotype with a prolonged lag phase and decreased cell yields for growth on both glucose and cellobiose. While the maximum endoglucanase activity and cellulase activity were not significantly changed, the endoglucanase activity and cellulase activity per cell were highly elevated. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR protein potentially acts as a repressor for some genes and as an activator for other genes. Overall, we established and demonstrated a method for manipulating chromosomal DNA in T. fusca that can be used to study the cellulolytic capabilities of this organism. Components of this method may be useful in developing genetic engineering methods for other currently intractable organisms.