As a global regulatory mechanism, carbon catabolite repression allows bacteria and eukaryal microbes to preferentially utilize certain substrates from a mixture of carbon sources. The mechanism varies among different species. In Pseudomonas spp., it is mainly mediated by the Crc-Hfq complex which binds to the 5' region of the target mRNAs, thereby inhibiting their translation. This molecular mechanism enables P. putida to rapidly adjust and fine-tune gene expression in changing environments. Hfq is an RNA-binding protein that is ubiquitous and highly conserved in bacterial species. Considering the characteristics of Hfq, and the widespread use and rapid response of Crc-Hfq in P. putida, this complex has the potential to become a general toolbox for post-transcriptional multiplex regulation. In this study, we demonstrate for the first time that transplanting the pseudomonal catabolite repression protein, Crc, into E. coli causes multiplex gene repression. Under the control of Crc, the production of a diester and its precursors was significantly reduced. The effects of Crc introduction on cell growth in both minimal and rich media were evaluated. Two potential factors - off-target effects and Hfq-sequestration - could explain negative effects on cell growth. Simultaneous reduction of off-targeting and increased sequestration of Hfq by the introduction of the small RNA CrcZ, indicated that Hfq sequestration plays a more prominent role in the negative side-effects. This suggests that the negative growth effect can be mitigated by well-controlled expression of Hfq. This study reveals the feasibility of controlling gene expression using heterologous regulation systems.
Keywords: Alkane monooxygenase; Attachment site; Genetic toolbox development; Medium chain fatty ester; Multiplex gene repression; Small RNA.
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