Gongliang Yu

Allelopathic mechanism of pyrogallol to Microcystis aeruginosa PCC7806 (Cyanobacteria): From views of gene expression and antioxidant system

Pyrogallol is a potent allelochemical on Microcystis aeruginosa, but its allelopathic mechanism is not fully known. In order to explore this mechanism, gene expressions for prx, mcyB, psbA, recA, grpE, fabZ under pyrogallol stress were studied, and activities of the main antioxidant enzymes were also measured. The results showed that expression of grpE and recA showed no significant change under pyrogallol stress, while psbA and mcyB were up-regulated at 4 mg L(-1). Both prx and fabZ were up-regulated even under exposure to 1 mg L(-1) pyrogallol concentration. The activities of superoxide dismutase (SOD) and catalase (CAT) were enhanced under pyrogallol stress. Levels of malodialdehyde (MDA) at 2 and 4 mg L(-1) pyrogallol were significantly higher than those of the controls. It was concluded that oxidant damage is an important mechanism for the allelopathic effect of pyrogallol on M. aeruginosa.

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.

Non-microcystin producing Microcystis wesenbergii (Komárek) Komárek (Cyanobacteria) representing a main waterbloom-forming species in Chinese waters

It is well known that several morphospecies of Microcystis, such as Microcystis aeruginosa (Kützing) Lemmermann and Microcystis viridis (A. Brown) Lemmermann can produce hepatotoxic microcystins. However, previous studies gave contradictory conclusions about microcystin production of Microcystis wesenbergii (Komárek) Komárek. In the present study, ten Microcystis morphospecies were identified in waterblooms of seven Chinese waterbodies, and Microcystis wesenbergii was shown as the dominant species in these waters. More than 250 single colonies of M. wesenbergii were chosen, under morphological identification, to examine whether M. wesenbergii produce hepatotoxic microcystin by using multiplex PCR for molecular detection of a region (mcyA) of microcystin synthesis genes, and chemical analyses of microcystin content by ELISA and HPLC for 21 isolated strains of M. wesenbergii from these waters were also performed. Both molecular and chemical methods demonstrated that M. wesenbergii from Chinese waters did not produce microcystin.

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.

First report on the allelopathic effect of Tychonema bourrellyi (Cyanobacteria) against Microcystis aeruginosa (Cyanobacteria)

The allelopathic effect of the cyanobacterium Tychonema bourrellyi against the cyanobacterium Microcystis aeruginosa is reported for the first time in this paper. The filtrate of T. bourrellyi CHAB663 culture showed strong inhibitory effect on M. aeruginosa NIES-843, but the inhibitory effect was weakened by shaking culture, and such results implied that the allelopathic effect was probably mediated by the volatile substances secreted by T. bourrellyi. β-Ionone was identified as a major ingredient in the volatile substances in the cultures of T. bourrellyi, and it may act as an important allelochemical responsible for this allelopathic activity. The filtrates of T. bourrellyi culture were shown to decrease the maximum electron transport rate (ETRmax) and elevate the reactive oxygen species (ROS) levels in the cells of M. aeruginosa NIES-843.

First report of microcystin production in Microcystis smithii Komárek and Anagnostidis (Cyanobacteria) from a water bloom in Eastern China

A water bloom sample collected from Lake Dishui in Shanghai was characterized. The morphological identification showed that Micorcystis wesenbergii and Micorcystis smithii were the main component of the bloom. Five strains of M. smithii were successfully isolated. Their 16S rRNA gene sequences based phylogenetic tree showed that the five strains of M. smithii intermixed with strains of other morphospecies in Microcystis. A fragment of mcy gene encoding for microcystin synthetase was detected in one of the five M. smithii strains (CHAB 2183), indicating its potential of microcystin production. High performance liquid chromatography analysis confirmed M. smithii CHAB 2183 to produce microcystin-RR as 1550 microg per gram dry weight cells. The present investigation, for the first time, reported the isolated strains of M. smithii and microcystin production from M. smithii.

Genetic analysis on Dolichospermum (Cyanobacteria; sensu Anabaena) populations based on the culture-independent clone libraries revealed the dominant genotypes existing in Lake Taihu, China

Lake Taihu has been severely eutrophied during the last few decades and dense cyanobacterial blooms have led to a decrease in phytoplankton diversity. The cyanobacterial blooms in Lake Taihu were mainly composed of unicellular colony-forming Microcystis and filamentous heterocystous Dolichospermum (formerly known as planktonic species of Anabaena). In contrast to that of Microcystis spp., the fundamental knowledge about diversity, abundance and dynamics of Dolichospermum populations in Lake Taihu is lacking. The present study was conducted to understand genotypic distribution, dynamics and succession of Dolichospermum populations in Lake Taihu. By sequencing 688 internal transcribed spacer (ITS) regions between the 16S and 23S rRNA genes of Dolichospermum, we were able to confirm that all the sequences were Dolichospermum rather than Aphanizomenon. 118 different genotypes were identified from the obtained sequences, and two genotypes (W-type and L-type) were found to dominate in the lake, representing 36.6% and 26.2% of the total sequences, respectively. These two dominant genotypes of Dolichospermum displayed the significant seasonal pattern. Stepwise regressions analysis revealed that water temperature was associated with the two dominant genotypes. The combined results implied the possible existence of ecotypes in bloom-forming cyanobacteria, probably triggered by water temperature in the lake.

Towards clarification of the inhibitory mechanism of wheat bran leachate on Microcystis aeruginosa NIES-843 (cyanobacteria): physiological responses

Wheat bran leachate (WBL) has been shown to have an inhibitory effect on Microcystisaeruginosa in this study. In order to explore the inhibitory mechanism of WBL on M. aeruginosa, physiological responses of M.aeruginosa NIES-843 under the WBL stress were studied. The expressions of six important genes related to the D1 protein of photosynthetic processes (psbA), synthesis of microcystins (mcyB), antioxidant protein peroxiredoxin (prx), synthesis of fatty acid (fabZ) and the repair of biological macromolecules (recA, grpE) were studied. Under the WBL stress, no significant change was found in expressions of both grpE and recA, but expressions of psbA, fabZ and prx were shown to be down-regulated, and slight up-regulated expression was found in mcyB. It was shown that oxygen evolution of M. aeruginosa NIES-843 was significantly depressed, and intracellular ATP contents became lower, after exposure to WBL. Similarly, maximum electron transport rates of photosynthetic activities decreased significantly, but intracellular reactive oxygen species levels boosted dramatically under the WBL stress, and cell lysis was observed. Therefore, it is suggested that photosynthetic systems and membranes were the potential targets of toxicity of WBL on M. aeruginosa, and the oxidative damage is an important mechanism explaining the inhibitory effect of WBL on M. aeruginosa.