Anticyanobacterial Effect Of L-lysine On Microcystis Aeruginosa

The excessive growth of cyanobacterial is caused by euthrophication,which has threatened aquatic ecosystems and the health of human.Therefore,effectives strategies to control and eliminate cyanobacterial blooms are urgently needed.L-lysine is one of the essential amino acids for microbial growth and has a significant algicidal effect on Microcystis species.In this paper,Microcystis aeruginosa,one of the bloom-forming cyanobacterial in freshwater worldwide,was selected to be the target for research.The effect and anticyanobacterail mechanism of L-lysine on M.aeruginosa was studied.Meanwhile,we also fully sequenced genomes of M.aeruginosa NaRes975.Based on the genome sequencing analysis,we selected the genes involved in polysaccharide synthesis and c-di-GMP metabolism.We tested their transcriptional activity and conducted heterologous expression of their encoding proteins,in order to explore the molecular mechanism M.aeruginosa cells in response to the effect of L-lysine.The main research contents and results are as follows:(1)The effects of L-lysine on M.aeruginosa were investigated from cell morphology,physiology,metabolism and molecular regulation.The results showed that L-lysine could efficiently inhibit the growth of M.aeruginosa at dosages above 5.0mg/L.But M.aeruginosa FACHB-905 takes an increasingly longer time than M.aeruginosa NaRes 975.The contents of protein and Chl-a were reduced at dosages above 0.5mg/L after 1d treatment and the photosynthetic system was also affected.Additionally,the MDA,O2-content and SOD activity increased within 1 d of treatment with 3.0,5.0,6.5,and 8.0 mg L-1 L-lysine,revealing that L-lysine induced oxidative stress in the cyanobacterial cells.The production of exopolysaccharide by M.aeruginosa also increased and the expression of polysaccharide synthesis genes was upregulated by 3.0 mg L-1 L-lysine after 3 d of treatment.These findings suggested that M.aeruginosa resisted damage by L-lysine using different strategies.According to SEM images,cyanobacterial cells were destroyed with>3.0 mg L-1 L-lysine treatment after 3 d.Additionally,for different strains,L-lysine has different effect.L-lysine has a better inhibitory effect on M aeruginosa and Anabaena.L-lysine could inhibit the growth of M.aeruginosa FACHB-905 cells and reduce the generation of extracellular MCs with 3.0 mg L-1 L-lysine treatment.(2)The genome sequencing of NaRes975 was performed using Illumina massively parallel sequencing platform(HiSeq 4000).The draft genome sequence of M.aeruginosa NaRea975 has been deposited at GenBank under the accession number MOLN00000000.The total size of the draft genome is 5,117,533-bp with a mean G+C content of 42.4%.The total of predicted protein-coding sequences(CDSs)was 5,217.(3)We examined on the expression of genes involved in polysaccharide synthesis and c-di-GMP metabolism.In the study,the genes were obtained by degenerate polymerse chain reaction.And then we performed the heterologous expression of the genes in E.coli BL21(DE3)and Rosetta gamiTM 2(DE3)plysS,using the pET-32a(+)as expressing vector.SDS-PAGE experiments showed that the gene for polysaccharide synthesis was failed to express although optimizing the concentration of IPTG and incubation temperature.The gene for c-di-GMP metabolism was expressed mainly as inclusion bodies in cell fragmentation.In conclusion,this study discusses the anticyanobacterial mechanism through investigating the inhibition of L-lysine on M.aeruginosa.And it provides basis for the application of L-lysine and enriches the approach towards prevention and mitigation of cyanobacteria excessive growth.In addition,the study also discussed the molecular regulation mechanism of M.aeruginosa cells when treated with L-lysine.Therefore it provides the expermental evidence for the possible involvement of signal regulation in cyanobacteria during treatment with L-lysine.