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Genomic Library Construction, mazEF Gene Clone from Microcystis Aeruginosa PCC 7820 and Its Function Research

Author: ShiZuo
Tutor: ZhangJun
School: Xiamen University
Course: Biochemistry and Molecular Biology
Keywords: Microcystis aeruginosa PCC 7820 toxin-antitoxin systems protein interaction
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 127
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Abstract


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 mazEFma.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 MazFma as a member of the growth inhibitor PemK-like protein family,which includes E.coli MazF.Similar with other TA modules,mazFma,coding for a 13-kDa protein,locates immediately downstream and overlaps by 4 nucleotides with the putative mazEma stop codon,mazEma encodes for 8.7-kDa protein.Through reverse PCR,more sequence information was known, including the promoter of mazEFma,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 mazEFma promoter is not strong or has no effect in E.coli lack of some factors.The MazFma overexpressed in E.coli repressed the cell growth and was neutralized by MazEma to relieve its toxic effect.And the interaction of MazEma and MazFma in vitro has been proved.Protein-protein interaction analysis illustrates that some proteins participate the effect of MazFma.And MazEma is a labile protein, degraded easily in the cell.Now we could not ensure the protein which degrades MazEma is the protease ClpAP,reported in E.coli,but this protein will degrade excessive MazEma.The different condition will influence the amount of MazEma and MazFma in the cell.This is the first report that demonstrates a physiological role for TA modules in cyanobabteria.

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