Study On The Function Of Redundant Cell Division Protein FtsZ Genes In Haloferax Mediterranei

Haloferax mediterranei DF50 exhibits a unique physiological mechanism for cell division.Through homology analysis,it has been discovered that multiple ftsZ genes exist in its genome.Currently,most research focuses on two dominant ftsZ genes,ftsZ1 and ftsZ2,which play key roles in cell division.The specific functions of other ftsZ genes have not been extensively studied and may either serve redundant purposes or function under specific conditions.This paper aims to investigate the impact of ftsZ gene deletion,individually or in combination,on bacterial cell morphology and the progression of cell division under different physiological and biochemical conditions.The specific research contents are as follows:（1）We constructed gene knockout plasmid vectors for Haloferax mediterranei DF50 and used homologous recombination to generate single and combined knockout strains for the four redundant ftsZ genes present in the genome of H.mediterranei DF50.This approach allows us to study the biological functions of the redundant ftsZ genes in H.mediterranei,such as their involvement in intracellular substance transport,stress response,cell wall synthesis,and stabilization of the cell division Z-ring.These knockout strains provide a feasible reference for investigating the different biological functions of the ftsZ redundant genes in H.mediterranei through gene deletion.（2）Four different conditions were selected to examine the impact of the knockout strains for H.mediterranei DF50 and its four redundant ftsZ genes on cell division.These conditions included potassium ion stimulation,varying salt concentrations,temperature variations,and cultivation in nutrient-limited media for fermentation.The growth curves were measured to analyze the preliminary effects of the absence of the four redundant FtsZ proteins on normal cell division.The experimental observations indicated that compared to the wild-type H.mediterranei DF50,the combination knockout strains ftsZ3、ftsZ4、ftsZ5 exhibited decreased optical density（OD）values at different cultivation times,particularly at 28°C,in a high-salt environment（25%salt concentration）,and under conditions of nitrogen and carbon source deprivation.The knockout of multiple redundant ftsZ genes hindered cell division in H.mediterranei DF50.（3）Through experimental design,we investigated the effects of the knockout of redundant ftsZ genes on the physiological and biochemical characteristics of H.mediterranei DF50 under conditions of relatively low temperature,high salt,and nutrient deprivation.The experimental results indicated that the four FtsZ proteins exhibited functional redundancy,but in the combination knockout strain ftsZ3,4,5under extreme conditions such as low temperature,the expression level of the remaining FtsZ6 protein was significantly downregulated.This suggests a certain degree of functional correlation among these four redundant ftsZ genes,and the knockout of multiple redundant ftsZ genes resulted in a significant decrease in the functionality of the remaining redundant ftsZ genes.Furthermore,the combined knockout of multiple redundant ftsZ genes also affected the expression of the major cell division proteins FtsZ1 and FtsZ2,thereby influencing bacterial growth and cell division activities.In a high-salt environment,the growth rate and cell count of the ftsZ3、ftsZ4、ftsZ5 combination knockout strain were significantly reduced,possibly due to the loss or silencing of the four redundant ftsZ genes,leading to decreased salt tolerance or hindered cell division.Under nutrient-deprived conditions,the expression levels of ftsZ1 and ftsZ2 were downregulated.Combined with the absence or silencing of redundant FtsZ proteins,this resulted in severe hindrance to cell division activities,and the bacteria tended to accumulate intracellular aggregates,leading to abnormal changes in the optical density(OD₆₀₀)values.The redundant ftsZ genes play a synergistic role in the process of cell division,possibly promoting normal cell division at different stages.Studying the mechanisms of these ftsZ genes in cell division provides a deeper understanding of the adaptation strategies of H.mediterranei to extreme conditions and its unique cellular division mechanism.In summary,this study reveals the functional roles of four ftsZ homologous genes,namely ftsZ3,ftsZ4,and ftsZ5,in addition to the main FtsZ protein-encoding genes ftsZ1 and ftsZ2,in H.mediterranei from a microbial genetics perspective.The research on the ftsZ3,ftsZ4,and ftsZ5 triple-knockout strains under low-temperature and high-salt conditions indicates a potential functional redundancy among the FtsZ3,FtsZ4,and FtsZ5 proteins,where the cellular physiological functions are compensated by the third FtsZ protein in the absence of any two genes.In the background strain H.mediterraneiΔftsZ3&ftsZ4&ftsZ5,the unsuccessful attempt to knock out the ftsZ6gene suggests that the FtsZ6 protein may possess similar functions as FtsZ3,FtsZ4,and FtsZ5.However,the available data are not sufficient to draw definitive conclusions at present.Obtaining more comprehensive combination knockout strain resources will help elucidate the function of the FtsZ6 protein and further confirm the complementary relationship between FtsZ3,FtsZ4,and FtsZ5.