Choi Jinlyung, Klingeman Dawn M, Brown Steven D, Cox Chris D
Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN 37996 USA.
Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996 USA.
Biotechnol Biofuels. 2017 Jun 24;10:163. doi: 10.1186/s13068-017-0849-2. eCollection 2017.
utilizes a wide variety of free and cellulosomal cellulases and accessory enzymes to hydrolyze polysaccharides present in complex substrates. To date only a few studies have unveiled the details by which the expression of these cellulases are regulated. Recent studies have described the auto regulation of the operon and determined that the -- gene cluster and nearby - gene cluster are co-transcribed as polycistronic mRNA.
In this paper, we demonstrate that the GlyR3 protein mediates the regulation of We first identify putative GlyR3 binding sites within or just upstream of the coding regions of and . Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative site in comparison to the site. Neither the putative site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the binding site, binding to the site was unaffected. In the presence of laminaribiose, in vivo transcription of the -- gene cluster increases, while expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription.
Together these results reveal a mechanism by which is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, is able to co-regulate both the and gene clusters in response to the availability of β-1,3-polysaccharides in its environment.
利用多种游离和纤维小体纤维素酶及辅助酶来水解复杂底物中存在的多糖。迄今为止，仅有少数研究揭示了这些纤维素酶表达的调控细节。近期研究描述了操纵子的自我调控，并确定--基因簇和附近的-基因簇作为多顺反子mRNA共同转录。
在本文中，我们证明GlyR3蛋白介导了的调控。我们首先在和的编码区域内或其上游鉴定出假定的GlyR3结合位点。使用电泳迁移率变动分析（EMSA），我们确定与位点相比，需要更高浓度的GlyR3才能有效结合假定的位点。假定的位点和随机DNA均未与GlyR3显著结合。虽然层二糖干扰了GlyR3与结合位点的结合，但与位点的结合不受影响。与不存在层二糖时相比，在存在层二糖的情况下，--基因簇的体内转录增加，而表达受到抑制，这与EMSA的结果一致。体外转录分析表明，GlyR3与层二糖的相互作用导致了观察到的体内转录模式。
这些结果共同揭示了一种机制，即GlyR3浓度低时表达，浓度高时受到抑制。通过这种方式，能够根据其环境中β-1,3-多糖的可用性共同调控和基因簇。
