Yongguang Jiang

Elucidating the toxicity targets of β-ionone on photosynthetic system of Microcystis aeruginosa NIES-843 (Cyanobacteria)

In order to explore the potential targets of toxicity of β-ionone on the photosynthetic system of Microcystis aeruginosa, the polyphasic rise in chlorophyll a (Chl a) fluorescence transient and transcript expression for key genes in photosystem II (PSII) of M. aeruginosa NIES-843 were studied. The EC₅₀ value of β-ionone on M. aeruginosa NIES-843 was found to be 21.23±1.87 mg/L. It was shown that β-Ionone stress can lead to a decrease in pigment content of M. aeruginosa NIES-843 cells, and that carotenoids were more sensitive to β-ionone stress than Chl a. The normalized Chl a fluorescence transients were slightly decreased at 6.67 and 10 mg/L β-ionone, but significantly increased at 15, 22.5 and 33.75 mg/L. There was no significant variation on transcript expression of psbA and psbO at a concentration of 6.67 mg/L β-ionone, but they were down-regulated at 22.5 mg/L. Ultrastructural examination by transmission electron microscopy indicated that the thylakoids were distorted, and the thylakoid membrane stacks began to collapse when M. aeruginosa NIES-843 was exposed to β-ionone at a concentration of 22.5 and 33.75 mg/L. Our results indicate that the reaction centre of PS II and the electron transport at the acceptor side of PS II are the targets responsible for the toxicity of β-ionone on the PS II of M. aeruginosa NIES-843.

Molecular Basis and Phylogenetic Implications of Deoxycylindrospermopsin Biosynthesis in the Cyanobacterium Raphidiopsis curvata

ABSTRACT New insights into the distribution and biochemistry of the cyanotoxin cylindrospermopsin (CYN) have been provided by the recent determination of its biosynthesis gene cluster (cyr) in several cyanobacterial species. Raphidiopsis curvata CHAB1150 isolated from China was analyzed for CYN analogues. Only 7-deoxy-CYN was detected in the cell extracts. The cyr gene cluster of R. curvata CHAB1150 was sequenced, and the cyr genes of this strain were found to have extremely high similarities (96% to 100%) to those from other nostocalean species. These species include Cylindrospermopsis raciborskii AWT205, Aphanizomenon sp. strain 10E6, and Aphanizomenon ovalisporum ILC-146. Insertion mutation was identified within the cyrI gene, and transcripts of cyrI and another functional gene cyrJ were detected in R. curvata CHAB1150. General congruence between the phylogenetic trees based on both cyr and 16S rrn was displayed. Neutral evolution was found on the whole sequences of the cyr genes, and 0 to 89 negative selected codons were detected in each gene. Therefore, the function of CyrI is to catalyze the oxygenation of 7-deoxy-CYN in CYN biosynthesis. The transcripts of the mutated cyrI gene may result from polycistronic transcription. The high conservation of the cyr genes may be ascribed to purifying selection and horizontal gene transfer.

Active and silent members in the mlr gene cluster of a microcystin-degrading bacterium isolated from Lake Taihu, China

The microcystin-degrading genes, mlr, are important participants in the degradation process of hepatotoxic microcystins for several bacterial species. However, their expression status during degrading microcystins is still unknown. In order to study this expression process, we isolated a novel microcystin-degrading bacterial strain, sequenced its mlr gene cluster and examined the expression of the mlrA gene at different concentrations of microcystin LR. The expression of mlrA increased slightly at 0.4 mg L⁻¹, and was significantly upregulated at 2.0 mg L⁻¹. Frameshift mutations were found in the mlrB* gene, and the mRNA of mlrB* could not be detected in the total RNA extracts of Novosphingobium sp. THN1. We conclude that mlrA is actively involved in the microcystin-degrading process, but mlrB* has lost its activity in this bacterial strain.

Variation of Microcystis and microcystins coupling nitrogen and phosphorus nutrients in Lake Erhai, a drinking-water source in Southwest Plateau, China

Lake Erhai is the second largest lake of Southwest China and an important drinking water source. The lake is currently defined as the preliminary stage of eutrophic states, but facing a serious threat with transfer into intensive eutrophication. The present study examined the dynamics of Microcystis blooms and toxic Microcystis in Lake Erhai during 2010, based on quantitative real-time PCR method using 16S rRNA gene specific for Microcystis and microcystin systhesis gene (mcy), and chemical analysis on microcystin (MC) concentrations. Total Microcystis cell abundance at 16 sampling sites were shown as an average of 1.7 × 107 cells l−1 (1.3 × 102–3.8 × 109 cells l−1). Microcystin LR (MC-LR) and microcystin RR (MC-RR) were the main variants. The strong southwesterly winds, anticlockwise circular flows and geographical characteristics of lake and phytoplankton community succession impacted the distribution patterns of Chl a and MC in the lake. The concentration of Chl a and MC and abundances of total Microsytis and MC-producing Microsystis (MCM) were shown to be positively correlated with pH, DO and TP, negatively correlated with SD, NO3-N, TN/Chl a and TN/TP, and not correlated with NH4-N, TN, dissolved total nitrogen (DTN) and water temperatures. When TN/TP decrease, Microcystis tended to dominate and MC concentrations tended to increase, suggesting that the “TN/TP rule” can be partially applied to explain the correlation between the cyanobacterial blooms and nutrients N and P only within a certain nutrient level. It is speculated that N and P nutrients and the associated genes (e.g., mcy) may jointly drive MC concentration and toxigenicity of Microcystis in Lake Erhai.

Sporadic distribution and distinctive variations of cylindrospermopsin genes in cyanobacterial strains and environmental samples from Chinese freshwater bodies

ABSTRACT Increasing reports of cylindrospermopsins (CYNs) in freshwater ecosystems have promoted the demand for identifying all of the potential CYN-producing cyanobacterial species. The present study explored the phylogenetic distribution and evolution of cyr genes in cyanobacterial strains and water samples from China. Four Cylindrospermopsis strains and two Raphidiopsis strains were confirmed to produce CYNs. Mutant cyrI and cyrK genes were observed in these strains. Cloned cyr gene sequences from eight water bodies were clustered with cyr genes from Cylindrospermopsis and Raphidiopsis (C/R group) in the phylogenetic trees with high similarities (99%). Four cyrI sequence types and three cyrJ sequence types were observed to have different sequence insertions and repeats. Phylogenetic analysis of the rpoC1 sequences of the C/R group revealed four conserved clades, namely, clade I, clade II, clade III, and clade V. High sequence similarities (>97%) in each clade and a divergent clade IV were observed. Therefore, CYN producers were sporadically distributed in congeneric and paraphyletic C/R group species in Chinese freshwater ecosystems. In the evolution of cyr genes, intragenomic translocations and intergenomic transfer between local Cylindrospermopsis and Raphidiopsis were emphasized and probably mediated by transposases. This research confirms the existence of CYN-producing Cylindrospermopsis in China and reveals the distinctive variations of cyr genes.

Variations of Growth and Toxin Yield in Cylindrospermopsis raciborskii under Different Phosphorus Concentrations

The bloom-forming cyanobacteria, Cylindrospermopsis raciborskii, is a producer of the cytotoxic cylindrospermopsin (CYN). In this study, the growth, toxin yield, and expression of CYN biosynthesis genes of C. raciborskii were examined under varying phosphorus (P) concentrations. The results show the cell number at 0.00 and 0.01 mg·L−1 P was significantly lower than that at higher P concentrations (≥0.5 mg·L−1). The chlorophyll a content, filament length, heterocyst, and akinete numbers at P ≤ 0.05 mg·L−1 were also significantly reduced. The intracellular and extracellular CYN concentrations and the extracellular proportions increased during the culture period, and larger values were observed at higher P concentrations. Total CYN content reached 45.34–63.83 fg·cell−1 and extracellular CYN proportion reached 11.49%–20.44% at the stationary growth phase. A significantly positive correlation was observed between CYN production and cell growth rate. Three cyr genes were expressed constantly even at P-deficient conditions. The transcription of cyr genes at P-replete conditions or after P supplementation increased from 1.18-fold to 8.33-fold. In conclusion, C. raciborskii may rapidly reorganize metabolic processes as an adaptive response to environmental P fluctuations. CYN production and cyr gene expression were constitutive metabolic processes in toxic C. raciborskii.

Congruence between mcy based genetic type and microcystin composition within the populations of toxic Microcystis in a plateau lake, China

Genetic diversity and differential microcystin contributions within the populations of toxic Microcystis in freshwater ecosystems have not been fully discussed. To illustrate this issue, we sequenced clone libraries of the first adenylation domain of mcyB (mcyBA1) from Microcystis populations in a plateau lake. Phylogenetic analysis revealed two divergent groups of McyBA1 with two closely related subgroups within Group I. While neutral evolution was indicated on the whole McyBA1 domain, 1-5 recombination breakpoints and several codons under positive or negative selections were found. Significant seasonal changes of McyBA1 diversity were observed. Group I composed the major proportion of the McyBA1 pool throughout the growing season in the whole lake. Group IA and Group II denominated Microcystis strains isolated from this lake were characterized by preference production of microcystin-RR (62-85%) and microcystin-LR (> 98%) respectively. We detected the intracellular microcystins in lake water and microcystin-RR was a main variant (mostly > 50%). In summary, McyBA1 subgroups were dominant within the population of toxic Microcystis and contributed the predominance of microcystin-RR in the lake. The differences of substrate preference in microcystin biosynthesis among groups were caused by neutral evolution and homologous recombination.

Re-evaluation of the diversity and distribution of diazotrophs in the South China Sea by pyrosequencing the nifH gene

Previous studies on the diversity and composition of nifH genes in the South China Sea (SCS) were mainly based on quantitative PCR and DNA clone methods. The pyrosequencing results of partial nifH gene fragments were used to study the spatiotemporal heterogeneity in composition and diversity of diazotrophs in the SCS. Seawater samples were collected throughout the upper ocean (<200-m depth) in different sites, as follows: the Pearl River Estuary; Taiwan and Luzon Straits (which are affected by the Kuroshio Current); a Trichodesmium bloom site; and two open ocean sites. Sample collection was conducted in four surveys from 2007 to 2010. A large dataset containing 217599 nifH reads from 27 samples was obtained. Putative diazotrophs in the SCS have reasonably low diversities, and the -proteobacteria and Trichodesmium were the two dominant nifH phylogenetic groups. No significant difference was observed among different regions and years. However, a significant difference existed among the samples obtained from different water depths. The results provide a broad and general view of the diversity and composition of the putative diazotrophs in the SCS.

Genotypes of ITS region of rRNA in Microcystis (Cyanobacteria) populations in Erhai Lake (China) and their correlation with eutrophication level

Previous studies on spatiotemporal changes of Microcystis genotypes have shown that the existence and succession of dominant genotypes always occur in eutrophicated freshwater bodies. However, few studies have focused on the correlation between genotype composition and eutrophication level. In the present study, clone libraries of the internal transcribed spacer (ITS) of rrn operon were sequenced from Microcystis populations in Erhai Lake, a subtropical plateau lake in the preliminary eutrophication stage. The genotype composition of the Microcystis populations was highly variable at spatiotemporal scales, and 473 ITS genotypes were identified from the 800 ITS sequences obtained. However, no significantly dominant ITS genotypes existed in the lake. Comparison of Erhai Lake with four major lakes in China, namely, Taihu, Chaohu, Gucheng, and Shijiu Lakes, showed that the Microcystis ITS genotypes and genetic diversity were negatively correlated with eutrophication level. Extensive comparison of the Microcystis ITS genotypes from waters worldwide revealed that 440 ITS genotypes were unique to Erhai Lake, and no obvious phylogenetic correlations can be detected among the dominant genotypes from different water bodies. The high genetic diversity of the Microcystis populations in Erhai Lake may have resulted from the effect of the early stage of eutrophication.