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Date of Award
Doctor of Philosophy (PhD)
The availability of nitrogen, as one of the main nutrients, is a key limiting factor that influences the growth of crop plants and food security. The biological process of nitrogen fixation (conversion of atmospheric nitrogen to ammonia) is a highly energy demanding process mediated by the nitrogenase enzyme complex which is sensitive to inactivation by oxygen. These stringent physiological conditions pose significant challenges to fulfil the long term goal of engineering nitrogen-fixing (diazotrophic) plants. All known nitrogen-fixing organisms are prokaryotes, distributed across bacterial and archaeal domains. Cyanothece sp. ATCC 51142 (Cyanothece 51142) is the most intriguing among the diazotrophic bacteria as it performs both nitrogen fixation and oxygen evolving photosynthesis, two seemingly incompatible cellular processes in a single cell by employing a protective mechanisms (temporal separation) and it has the most efficient nitrogenase: highest activity for the longest period. However, the regulatory components and the mechanism of nitrogen-fixation of Cyanothece 51142 nif cluster remain unknown due to the lack of sufficient genetic tools in Cyanothece 51142.
This work presents the characterization of the specific binding of the putative cyanobacterial nitrogen fixation regulator of the unicellular Cyanothece 51142 (named CnfRCt) to the bidirectional promoter region of the nif cluster. Furthermore, we demonstrated the functional modularity of the identified transcriptional regulatory components in a heterologous host. First, we identified six binding sites containing previously unspecified, conserved nucleotides adjacent to the consensus TGAGT sequence in the intergenic region between nifB and nifP using DNase I footprinting. We further demonstrated the transcriptional activation of this bidirectional promoter region by CnfRCt using in vivo reporter assays in a heterologous bacterial host. We discovered that all binding sites enhanced the promoter activity, and all of them are required for maximal activation for CnfRCt. Moreover, the activation by CnfRCt requires other essential A/T- and G/C-rich inverted repeat motifs. These motifs are required for the promoter recognition by the E. coli RNA polymerase (RNAP), and possibly responsible for DNA bending. Furthermore, we studied the effect of CnfRCt on the expression of key nif genes and the nitrogenase activity in the previously engineered Synechocystis 6803 containing the minimal Cyanothece 51142 nif cluster in a synthetic replicative plasmid pSL2397. Finally, we explored the potential regulation of the bidirectional promoter and CnfRCt by other transcription factors that could be associated with the nitrogen fixation process in Cyanothece 51142. Taken together, this work shows that the transfer of a key transcriptional regulator of the key bidirectional promoter of the Cyanothece 51142 nif cluster could be functional in a non-nitrogen fixing bacteria.
Costas D. Maranas, Tae Seok Moon, Himadri Pakrasi, Yinjie Tang,
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