Studies On The Application Of Green Fluorescence Protein ECGP123 And Essentiality Of HerA-nurA Paralogs In Sulfolobus Islandicus

Green fluorescent protein （GFP） serves as a powerful fluorescent marker in the study of eukaryotic and prokaryotic cellular and molecular biology for gene expression and protein localization. Most archaea live in the harsh environments. The optimal living condition of the Crenarchaeon Sulfolobus islandicus is 75-80℃ and pH 2-3, in which most GFP would denature due to the high temperature and low pH and thus could not be utilized in S. islandicus cells. Among many GFP mutants, eCGP123 （enhanced consensus green protein variant 123） exhibits extreme thermostability, acid stability and reversible photoswitching. It has been applied in study on the effect of various type IV flagella component-deleting mutants on the formation of biofilm in S. acidocaldorius. However, the application of eCGP123 in cellular localization of archaeal proteins has not been reported.ATPase/helicase Her A （SiRe₀064） and nuclease NurA （SiRe₀061） are involved in S. islandicus DNA double-stranded breaks （DSBs） repair. They process dsDNA together with Mre11-Rad50 complex, forming a DNA with 3’-overhang. She et al. and those in our laboratory attempted to knockout the four genes in one opron, mrell, rad50, herA and nurA but failed, indicating that they are essential for cell viability. Besides the classical herA-nurA, there are 5 pairs of herA and nurA paralogs in S. islandicus genome. SiRe₀017, SiRe₀095, SiRe₀566, SiRe₀581 and SiRe₁531 are paralogs of herA, while SiRe₀014, SiRe₀094, SiRe₀565, SiRe₀582 and SiRe₁533 are paralogs of nurA. So far, their essentialities and in vivo functions are unknown.In this thesis, in order to study the application of eCGP123 as a reporter for protein cellular localization and the essentialities of herA and nurA paralogs in S. islandicus, we constructed eCGP123 fusion protein overexpression strains and herA-nurA paralog deletion mutants using the well-developed genetic system of S. islandicus. Their growth curves and cell morphology were analyzed.Firstly, the codon of eCGP123 gene was optimized and the gene was synthesized. eCGP123 expressed and purified from E. coli was greatly thermostable. And the E. coli cells exhibited green fluorescent by microscopy. eCGP123 and its fusion protein eCGP123-lacS were also expressed in S. islandicus, which demonstrated that eCGP123 is thermostable and acid-tolerant. Then, proteins with different functions were fused at the C-terminal of eCGP123 for studying their cellular localization.In E. coli, FtsZ is a cell division protein. As expected, we found that eCGP123-FtsZ mainly localized at the mid-cell. In S. islandicus, eCGP123 was evenly distributed in cytoplasma. After fused with UpsE, a secretion ATPase anchored in the cell membrane, eCGP123-UpsE was still evenly distributed in cytoplasma. eCGP123-PCNA1 exhibited 1 to 4 foci in cells. eCGP123-SiRe 1200-C-His localized at mid-cell, further confirming the previous result that it may be involved in cell division. eCGP123-SiRe 1388-no-His was distributed at the mid-cell and cell periphery, indicating that SiRe₁388 might also participate in cell division and be anchored in cell membrane. eCGP123-SiRe 1550 localized at one certain area in S. islandicus cells. The growth curves and cell morphology analysis showed that the growths of eCGP123-SiRe₁200, eCGP123-SiRe₁388, and eCGP123-SiRe₁550 were affected and cell sizes became enlarged. Taken together, eCGP123 can be used for protein localization. But there is still some trouble that would limit its application, such as low fluorescent signal, easily quenching, the effect of protein expression level on localization, and small size （1-2 μm in diameter） of Sulfolobus cells.In addition, we attempted to knockout the pairs of herA-nurA paralogs by marker replacement, and analyzed their essentiality in S. islandicus. Through screening on MSCV plate, we got SiRe₀014-SiRe₀017, SiRe₀094-SiRe₀095, SiRe₀581-SiRe₀582 and SiRe₁531-SiRe₁533 deletion mutants, respectively. Growth experiments and microscopy revealed wild-type growth and a normal cellular phenotype for all deletion mutants, suggesting that they are non-essential genes. However, we could not obtain a pure △SiRe₀565-SiRe₀566 strain, indicating that they might be essential for S. islandicus.