Genetic Analysis Of ESCRT-? Homologues In Sulfolobus Islandicus

Cell division is one of the most fundamental processes for all the life.The cell division mechanism differs between bacteria,archaea,eukarya,plants,and animals.Sulfolobus have mixed features of bacteria and eukarya,and they share similarities with eukarya in replication,transcription,and translation,however,the mechanism of Sulfolobus is simpler than eukarya.Rencently,cell division in Sulfolobus was found to resemble the counterpart of eukarya.It is important that study of the cell division mechanism in Sulfolobus would provide a simplified model and futher understanding of the cell division mechanism in eukaryotes.We kown little about how archaea cells divide currently,and the exploration of archaea cell division just at the very beginning.Recent studies have shown that three proteins,CdvA,CdvB and CdvC?cell division protein A,B and C?encoded by the hyperthermophilic arachaea Sulfolobus are involved in cell division.Among them CdvA is an archaea-specific protein.CdvB and CdvC are the homologues of the eukaryotic ESCRT-??endosomal sorting complex required for transport ??and vacuolar protein sorting 4?Vps4?,respectively.Intriguingly,Sulfolobus has other three ESCRT-? homologues besides CdvB,they are Saci₀451?SiRe₁550?,Saci₁416?SiRe₁200?,and Saci₁601?SiRe₁388?.Among them,Saci₀451?SiRe₁550?,Saci₁416?SiRe₁200?show cell periodic transcription pattern,and we can infer they have a potential role in Sulfolobus cell division,however the relationships between them and Vps4 have not been fully eucidated.And the roles and mechanisms of them in cell division are obscure.In this study,we utilized the genetic analysis system in Sulfolobus islandicus,and try to construct the knoukout type and/or the overexpression strain of SiRe₁200,SiRe₁550,and SiRe₁388,and conducted genetic analysis of the strains.Immunofluoresence experiment was also used to identify the location of the proteins.We investigated the in vivo roles of the three ESCRT-? protein homologues in S.islandicus through the above approaches and dissected their specific roles and mechanisms in archaeal cell division.Firstly,we overexpressed the mutant protein SiRe₁200?C.Sulfolobus islandicus was transformed with an empty vector,or a vector containing the mutant protein SiRe₁200?C.We found that overexpression strain of SiRe₁200?C grew as fast as the control before OD600 reached 0.3.Interestingly,it then began to grow apparently slowly afterwards.Another interesting observation of S.islandicus/pSeSD-SiRe₁200?C induced by arabinose was the appearence of a large number of enlarged cells,with a highest ratio of about 50%.The cell size differed significantly.The cell membrane was wrinkled and was not as smoth as the control.At the final stage,enlarged cells of 3-fold larger than the control appeared.Further we observed eukaryotic intercellular bridge-like structure in S.islandicus/pSeSD-SiRe₁200?C induced by arabinose,which is apparently cell division defects.It seemed that overexpression of SiRe₁200?C leading to the cells growing apparently slowly with different size and most of which were enlarged implied cell division defects.Thus we demonstrate SiRe₁200 may play a role in cell division.We also investigated the role of the other two ESCRT-? homologs,SiRe₁550 and SiRe₁388.We use pryEF-lacS selection marker and the double-crossover strategy to delete the genes.We had difficulties to knockout SiRe₁550.We always obtained a mixture strain of the wild type and the knonkout type,and the percentage of the wild type cells is 1%.The difficulty in SiRe₁550 deletion hints the importance of SiRe₁550.We observed that 3-6 cells were in toruloid aggregation morphology in S.islandicus/"?SiRe₁550",and 30%of the cells were in aggregation.It may imply a link between SiRe₁550 and cell division,and deletion of SiRe₁550 in most cells may lead to a disorder in cell division.As to SiRe₁388,the same strategy with SiRe₁550 was used.We obtained the knockout type of SiRe₁388,indicating SiRe₁388 is not essential.Cells became obviously aggregated of S.islandicus/?SiRe₁388 when cultured for 36 hours,and about 30 cells were in aggregation.We have not found out the relation between SiRe₁388 and cell division.To better understand the role SiRe₁200 playing in Sulfolobus,we also investigated the localization of SiRe₁200 in S.islandicus/pSeSD-SiRe₁200 by immunofluorescence assay,and the strain was induced by sucrose.We observed in several cells SiRe₁200 localized in the middle of segregated nucleoids which implying a cell in division phase,and this localization implies that SiRe₁200 play a role in Sulfolobus cell division.