| As an ancient photosynthetic microorganism,cyanobacteria can survive in ecologically diverse habitats due to their capacity to rapidly respond to environmental changes through complex signaling networks,including using c-di-GMP to regulate physiology or metabolism.In recent years,the occurrence of cyanobacterial blooms appears to be increasing because of environmental factors,such as continued eutrophication,rising atmospheric CO2 concentrations,and global warming.The proliferation of cyanobacteria in eutrophic water bodies cause harmful blooms thus has become the most common environmental problem worldwide.Microcystis is one of the most prevalent bloom-forming cyanobacterial genera and globally distribute in freshwater ecosystems.Microcystis is able to form colonial aggregates covered by EPS mucilage and control its buoyancy by the GV to absorb light and nutrients more efficiently.During blooming,large colonial aggregates form a scum floating on the surface of water bodies,which impair aquatic ecosystems and cause deterioration of water quality.Microcystis is a genus containing a great deal of genetic diversity.Some strains of Microcystis can synthesize a variety of toxic NRP cyanopeptides,such as hepatotoxic MCs,which pose a risk to humans and other animals.Therefore,it is necessary to explore the mechanism of Microcystis colonies aggregation for understanding the bloom-forming mechanism and controling Microcystis blooms reasonably.Currently,there are few studies on c-di-GMP signaling in Microcystis.The researches on relationship of aggregation morphology,physiological structure and response mechanism of microcystis cells are scattered.And a few studies have addressed the distribution of NRP synthesis genes and it is still unclear about the evolutionary history of NRP synthesis genes encoded by Microcystis,as well as the diversity and toxicity and enzyme-inhibiting activities of NRP produced by Microcystis.Based on this,comparative genomic analysis was performed in this study,which mainly focused on the distribution,evolutionary history,and diversity of c-di-GMP metabolism and regulation genes,as well as NRP gene clusters in Microcystis through bioinformatics approaches.And a variety of detection methods were used to explore the physiological response mechanism of M.aeruginosa cell aggregation.This study provides a physiological and genetic basis to enhance our understanding of Microcystis environmental adaption,bloom-forming mechanism and harmfulness caused by Microcystis blooms.The main contents and conclusions are as follows:(1)The genome sequencing and functional gene analysis of M.aeruginosa CHAOHU 1326 were conducted.The genome of M.aeruginosa CHAOHU 1326 had a total size of 5.27 Mb with a G+C content of 42.53%.In total,4,617 CDSs were identified from CHAOHU 1326.RNA-coding genes encoded by M.aeruginosa CHAOHU 1326 consist of 46 tRNA genes,4 sRNAs,and a set of 5S-16S-23S rRNA genes.Genome sequence analysis revealed that a cluster of twelve genes predicted to be involved in GV synthesis is encoded by M.aeruginosa CHAOHU 1326.Strain CHAOHU 1326 carries 92 genes involved in EPS synthesis and 3 genes involved in cdi-GMP metabolism.M.aeruginosa CHAOHU 1326 is a toxic strain with potential toxicity as genes involved in AER,CPT,and MC synthesis were identified in its genome.(2)The evolutionary history of the Microcystis genome,the distribution of the c-diGMP control module in Microcystis and the diversity of the synthesis genes and nonribosomal cyanopeptides were analyzed through in silico analyses.The number of CDSs contained by Microcystis are proportional to the size of Microcystis genomes.ANI and phylogenetic analysis of the Microcystis genome revealed that they have high similarity at the nucleotide level.The pan-genome analysis revealed that Microcystis have an open pan-genome and a conserved core genome.Propensity for gene loss analysis revealed that most of genes involved in c-di-GMP signaling are stable in Microcystis strains.Bioinformatics and structure analysis of c-di-GMP signal-related domains revealed that they all possess essential conserved amino acid residues that bind the substrate.AER,CPT,and MC biosynthesis gene clusters are the most frequently observed in the Microcystis genomes.Phylogenomic affiliation analysis of NRPS gene cluster profiles indicates that Microcystis phylogenomic marker genes and nonribosomal cyanopeptide synthesis genes have coevolved during evolution.Substrate and structure prediction indicates varied substrates can be activated by the Adomains and the nonribosomal cyanopeptides may have different compositions thus to have toxicity and inhibitory activities.Selection analysis demonstrates that all Adomains encoded by aer,mcn,and mcy gene clusters are subjected to purifying selection,and positive selection has acted on some residues in core motifs and substrate binding pockets of the A-domains.(3)The relationship among the morphology of M.aeruginosa strains,EPS and GV was investigated.Morphological analysis showed that the aggregated CHAOHU 1326 cells possess massive GVs and were encased by a thick extracellular layer,whereas the unicellular FACHB-925,FACHB-940 and FACHB-975 cells only had a little secretion on their surfaces and did not contain GV.CHAOHU 1326 produced a significantly higher amount of EPS than other unicellular morphology strains during growth.All the key EPS genes encoded by CHAOHU 1326 all showed transcriptional activity,while some of the genes were non-transcribed in M.aeruginosa strains FACHB-925 and FACHB-940.In addition,all of the predicted gvp genes were expressed in M.aeruginosa CHAOHU 1326.Colonial morphology and suspended growth of CHAOHU 1326 might be benefit from the strong ability of EPS synthesis and massive GVs.(4)The intracellular response of M.aeruginosa to L-Arg was investigated.The results of transcriptome sequencing revealed that L-Arg stimulated the up-regulation of expression levels of genes involved in nitrogen metabolism,carbon fixation,arginine metabolism,NRP and GV synthesis in M.aeruginosa,and also affected the expression of genes related to various photosynthetic pigments and photosystems.The physiological indexes of M.aeruginosa strains under different L-Arg concentrations were monitored.The results showed that the low concentrations(0.01,0.05 and 0.1 mM)of L-Arg have not affected cell division and growth of M.aeruginosa strains but promoted the MC synthesis.Whereas high concentration(0.5 and 1 mM)of L-Arg significantly inhibited the growth of M.aeruginosa strains,significantly promoted the EPS synthesis,cell aggregation and biofilm formation,and also stimulated CHAOHU 1326 to release MC.The variation of intracellular Chla and protein contents of M.aeruginosa strains are consistent with changes in cell density,which reflected the growth of M.aeruginosa strains.It was shown by RT-qPCR assay that L-Arg affected the expression levels of genes involved in EPS synthesis,MC synthesis and c-di-GMP metabolism.(5)Overexpressed transformants of M.aeruginosa for c-di-GMP metabolism gene hdp,downstream effector cellulose synthesis gene celA and EPS biosynthesis gene bcsA in M.aeruginosa were constructed.Based on different strategies,expression plasmids pRL1342-hdp,pGEM-T-hdp,pGEM-T-bcsA,and pGEM-T-celA were constructed and transferred into the M.aeruginosa CHAOHU 1326 cells by electroporation and achieved the transformants HDP-1326,BCSA-1326 and CELA1326,respectively.The overexpressed transformants were screened out through resistance selection and PCR verification. |
PDF Full Text Request