|Other Abstract||Microcystis is a group of unicellular cyanobacteria distributed all over the world and most frequently occur in freshwater lakes and ponds. In summer, massive production of Microcystis results in the outbreak of waterblooms, causing deterioration of water environments. In Microcystis cells, a special hollow cylindrical structure, gas vesicle, facilitates buoyancy and enables cells to perform vertical migration in water column and gain favorable growth conditions, consequently, plays an important role in the outbreak of Microcystis waterblooms. In this study, gas vesicle gene clusters from various Microcystis strains were compared, their phylogenetic relationship and differences in gene expression were investigated. A preliminary study on the regulation of gvp gene transcription in a strain of Microcystis was also conducted.
The main researches and results are in four parts:
(1) The sequence of the gvp (gas vesicle protein) gene cluster from Microcystis sp. FACHB854. The genomic library of FACHB854 was constructed and a clone carrying gvpA-gvpC gene was identified. Based on the sequence of this region and the published sequence of Microcystis PCC 7806 while this study was going on, the whole sequence of the 7.5-kb gene cluster was finally obtained using regular PCR, inverse PCR and sequencing. The gene cluster contains 11 genes, gvpA having two copies. Southern blot hybridization showed that only one gvp cluster is present in the genome of FACHB854.
(2) The structural types, phylogenetic relationships and coordinate regulation of gvpA-gvpC regions in Microcystis. By comparing the sequences of the gvpA-gvpC regions from 8 Microcystis strains, gvp clusters were classified into four structural types: 2gvpA+gvpC (3), 2gvpA+gvpC (4), 3gvpA+gvpC (4) and 4gvpA+gvpC (3). The phylogenetic trees based on partial rbcLX, intergenic sequences between gvpA copies or gvpA and gvpC, gvpC gene showed that the evolution of gvp cluster was basically consistent with the phylogeny of Microcystis. Measurements by electron microscopy confirmed that the diameter of gas vesicles in Microcystis was correlated with the length of GvpC protein, but not to the copy number of gvpA.
(3) A preliminary study on the transcriptional regulation of gvpA and gvpC genes. The effects of various environmental factors, including temperature, light intensity, nitrate concentration and pH value, on the buoyancy of FACHB930 were investigated in BG11 medium, and the results showed that the buoyancy of this Microcystis strain was apparently regulated by the initial pH value. Half-quantitative and quantitative RT-PCR both showed that transcriptional levels of 3 gvpA and gvpC genes changed coordinately with the initial pH value of the culture.
(4) A miniature transposable insertion element in Microcystis sp. FACHB 854. A 186-bp miniature insertion element, ISM854-1, was found in FACHB 854, with imperfect terminal inverted repeats, target direct repeats but without any transposase-encoding capacity. Southern blot hybridization showed that ISM854-1 and homologues were present with more than 10 copies in the genome of FACHB 854, but none or few in other Microcystis strains. More ISM854-1 copies were obtained employing inverse PCR, all located at 8-bp long and AT-rich target sequences. A variant of ISM854-1, denoted ISM854-1A, may transpose in pairs in a structure like a composite transposon.
The main conclusions of this study are:
(1) Gas vesicle gene clusters were classified into four structural types: 2gvpA+gvpC(3), 2gvpA+gvpC(4), 3gvpA+gvpC(4) and 4gvpA+gvpC(3), in which numerals before gvpA are copy numbers of tandem gvpA genes and those in the parentheses are numbers of conserved repeating units (33RR) in GvpC proteins.
(2) The transcriptional levels of 3 gvpA copies and gvpC gene were regulated coordinately in the strain FACHB930.
(3) A non-autonomous transposible miniature insertion element was found in a cyanobacterium.|