Genomic Library Construction, MazEF Gene Clone From Microcystis Aeruginosa PCC 7820 And Its Function Research

Microcystis aeruginosa is cosmopolitan,usually harmful,and one of the best studied genera of the cyanobacteria.It is coccoid,unicellular and present in natural waters.These cyanobacteria can flourish in aquatic environments to produce blooms that bring toxin.This toxin hazards human and animal health at environmentally-encountered concentrations.Toxin-antitoxin(TA) gene pairs,also known as addiction modules,have been implicated in stress management and survival by bacteria.They are two gene operons that encode an unstable antitoxin,which autoregulates expression of the operon,and a stable toxin,which is neutralized by forming a complex with the antitoxin.The most studied of these modules is Escherichia coli mazEF,in which mazF encodes a stable toxin,MazF,and mazE encodes a labile antitoxin,MazE,which prevents the lethal effect of MazF.In our study,we identified and characterized a putative TA pair in Microcystis aeruginosa PCC 7820,designated mazEF_ma.The nucleotide sequences were acquired from a genome library constructed in our lab,and have little identity with the E.coli mazEF,though NCBI searches placed MazF_ma as a member of the growth inhibitor PemK-like protein family,which includes E.coli MazF.Similar with other TA modules,mazF_ma,coding for a 13-kDa protein,locates immediately downstream and overlaps by 4 nucleotides with the putative mazE_ma stop codon,mazE_ma encodes for 8.7-kDa protein.Through reverse PCR,more sequence information was known, including the promoter of mazEF_ma,which is different from that of E.coli mazEF.The expected "alternating palindrome" was not found.The low level of GFP expression suggests that the mazEF_ma promoter is not strong or has no effect in E.coli lack of some factors.The MazF_ma overexpressed in E.coli repressed the cell growth and was neutralized by MazE_ma to relieve its toxic effect.And the interaction of MazE_ma and MazF_ma in vitro has been proved.Protein-protein interaction analysis illustrates that some proteins participate the effect of MazF_ma.And MazE_ma is a labile protein, degraded easily in the cell.Now we could not ensure the protein which degrades MazE_ma is the protease ClpAP,reported in E.coli,but this protein will degrade excessive MazE_ma.The different condition will influence the amount of MazEma and MazF_ma in the cell.This is the first report that demonstrates a physiological role for TA modules in cyanobabteria.