Establishment Of A Novel Gene Knockout Scheme And Genetic Analysis Of Pcna Gene In Sulfolobus Islandicus

PCNA (proliferating cell nuclear antigen) play an important role in many essential celluar processes, such as DNA repliacation, DNA repair, DNA recombination and cell cycle control. Organisms belonging to the Crenarchaeota lineage contain three PCNA subunits while the members in Euryarchaeota have only one PCNA as for Eukarya. Sulfolobus islandicus which is classified as Crenarchaeota lineage is being used as an ideal model for studying hyperthermophilic archaeal biology and it is found that S. islandicus genome contains three PCNA homologues (PCNA1, PCNA2 and PCNA3) encoding by pcnal, pcna2 and pcna3 gene respectively. Because of lack of powerfull Sulfolobus genetic system, the research of PCNA is just limited to the physiological and biochemical characterization in vitro while the genetic study of PCNA is far lagged. Based on the previous work in our lab, we further developed the genetic system, in which used the double mutant E233S as a host strain and pyrEF together with lacS as selectable markers. And then, the pcna genes from S. islandicus were conducted preliminary genetic analysis.In order to investigate the mechanism of Sulfolobus PCNA, we sought to generate pcna deletion mutants by taking adavantage of constructing different types of pcna knockout plasmids based on pyrEF and lacS selectable markers and then transforming into the host strain S. islandicus ApyrEFAlacS (E233S). However, we failed to get positive transformants with two conventional knockout methods, i.e.allelic replacement (AR) and marker replacement and looping out (MRL). Therefore, a novel knockout scheme denoted the marker insertion and target gene deletion (MID) was developed with which transformants were obtained by each pMID-pcna plasmid. According to the PCR analysis, we found that pcna mutant allele exsit in the pMID-pcna transformants all the time. Considering that pcna deletion mutants (Apcna) will survive in the presence of 5-Fluoroorotic-Acid (5-FOA) while pMID-pcna transformants can be selectively killed, mutant propagation assay was established. We incubated the pMID-pcna transformants in the liqud rich medium containd 5-FOA, and then identify whether Apcna cells can propagate during the counter selection enrichment by means of semi-quantitative PCR analyses for the deleted pcna gene allele.It was found that the proportion of Apcna cells in the culture maintained about 1% in the whole stage of mutant propagation assay, which indicated that pcna genes are absolutely required for the host growth. This is the first report to demonstrate microbial gene essentiality by assessing mutant propagation rather than colonization.We have also profiled a comparative studies on expression distinction of three S. islandicus pcna genes from transcriptional and protein level respectively. qRT-PCR assay revealed that the mRNA abundance of each pcna gene is almost the same at different growth phases (OD600=0.2,0.5,0.8,1.1) of S. islandicus. However, Western-blot analysis showed that the protein amounts of each PCNA have significant differences and they have a similar tendency as well. PCNA3 is the most abundant among the three PCNAs while that of PCNA2 is the lowest in cells. The disparities of protein amounts about three PCNAs may indicate different forms of functional PCNA trimeric rings exsit in vivo for S. islandicus.In the following work, we put the pcna genes under the control of araS promoter and constructed a series of PCNA expression vectors with or without C-terminal His tag based on the Sulfolobus expression plasmid pSeSd. And then, these plasmids were transformed into the host strain E233S, generating the PCNA overexpression strains. Western-blot analysis revealed each PCNA can be successfully overexpressed in S. islandicus. In order to know whether the overexpression of PCNA will exert an influnce on the cell or not, the growth curves were tested in the inducible medium (ACVy) and uninducible medium (GCVy). The results showed that the growth rate of PCNA overexpression strains had no difference compared to the wild type in GCVy medium. Surprisingly, in ACVy medium, once the PCNA2 was overexpressed, Sulfolbous cells would exhibit a delayed growth significantly while PCNA1 and PCNA3 overexpression strains not. Further research conducted by Flow cytometry displayed that the overexpression of PCNA2 in vivo would not have any affect on DNA replication although it will cause Sulfolobus cells to die to some exent.Finally, the novel gene knockout strategy MID has also been used to study the nucleotide excision repair and ligh repair related genes including xpb, xpd, xpf, xpg, bax1 and phrB. At present, we have constructed xpb1, xpb2, xpd, xpf, bax1 and phrB deletion mutants respectively and identified that xpg is unknocktable as well. The genetic analysis of these deletion mutants will be carried out in the following work, which will reveal the mechanism of nucleotide excision repair and ligh repair in archaea.