Physiological and thylakoid ultrastructural changes in cyanobacteria in response to toxic manganese concentrations.

In this study, two cyanobacterial strains (morphologically identified as Microcystis novacekii BA005 and Nostoc paludosum BA033) were exposed to different Mn concentrations: 7.0, 10.5, 15.7, 23.6 and 35.4 mg L-1 for BA005; and 15.0, 22.5, 33.7, 50.6, and 76.0 mg L-1 for BA033. Manganese toxicity was assessed by growth rate inhibition (EC50), chlorophyll a content, quantification of Mn accumulation in biomass and monitoring morphological and ultrastructural effects. The Mn EC50 values were 16 mg L-1 for BA005 and 39 mg L-1 for BA033, respectively. Reduction of chlorophyll a contents and ultrastructural changes were observed in cells exposed to Mn concentrations greater than 23.6 and 33.7 mg L-1 for BA005 and BA033. Damage to intrathylakoid spaces, increased amounts of polyphosphate granules and an increased number of carboxysomes were observed in both strains. In the context of the potential application of these strains in bioremediation approaches, BA005 was able to remove Mn almost completely from aqueous medium after 96 h exposure to an initial concentration of 10.5 mg L-1, and BA033 was capable of removing 38% when exposed to initial Mn concentration of 22.5 mg L-1. Our data shed light on how these cyanobacterial strains respond to Mn stress, as well as supporting their utility as organisms for monitoring Mn toxicity in industrial wastes and potential bioremediation application.

pH-dependent gas vesicle formation in Microcystis.

In lakes with seasonal cyanobacterial blooms, the pH fluctuates from slightly above 7 to around 10. In this study, we found that the abundance of gas vesicles in Microcystis species, in parallel to the buoyancy of cells, increased in response to elevation of the extracellular pH. Within 48 h after pH upshift, gas vesicle protein genes (gvp) were upregulated at both mRNA and protein levels due to reduced decay of gvp transcripts. The effect of pH on GvpC level was basically unaffected by inorganic carbon availability. This is the first report that long-term pH range plays a role in controlling gas vesicle formation in certain Microcystis species.

The combined effects of UV-C radiation and H2O2 on Microcystis aeruginosa, a bloom-forming cyanobacterium.

In order to get insight into the impacts of UVC/H2O2 on Microcystis aeruginosa, physiological and morphological changes as well as toxicity were detected under different UVC/H2O2 treatments. In the presence of sole UVC or H2O2, the net oxygen evolution rate decreased significantly (p<0.05) since activity of photosystem II (PSII) was inhibited. Meanwhile, increase of intracellular reactive oxygen species (ROS), degradation of microcystin (MC) and ultrastructure destructions were observed. Under sole UVC treatment, no changes happened in the activity of photosystem I (PSI), but the degradation of D1 protein was observed. Under sole H2O2 treatment, an increase of malondialdehyde, aggregation of D1 protein and deformation of the thylakoid membrane were observed. ROS content under H2O2 treatment was about 5 times than that under UVC treatment. Combined use of UVC and H2O2, as well as 20mJcm(-2) UVC and 60µM H2O2, showed high synergetic effects. Obvious damage to membrane systems, the marked degradation of MC and inhibition of the photosystems were observed. It could be deduced that UVC worked on intracellular membrane components directly and the damaged oxygen-evolving complex, which was followed by the D1 protein degradation. H2O2 oxidised the membrane lipids via an ROS-mediated process, with thylakoid injury and the aggregation of D1 protein being the lethal mechanisms, and both PSII and PSI being the attacking targets. With regard towards the effective inactivation of M. aeruginosa and high removal of MC, UVC/H2O2 proposed a novel practical method in controlling cyanobacterial blooms.

Structure of the gas vesicle protein GvpF from the cyanobacterium Microcystis aeruginosa.

Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8-14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF from Microcystis aeruginosa PCC 7806 is reported at 2.7 Šresolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an α+ß class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each other via interactions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.

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

In order to explore the potential targets of toxicity of ß-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 ß-ionone on M. aeruginosa NIES-843 was found to be 21.23±1.87 mg/L. It was shown that ß-Ionone stress can lead to a decrease in pigment content of M. aeruginosa NIES-843 cells, and that carotenoids were more sensitive to ß-ionone stress than Chl a. The normalized Chl a fluorescence transients were slightly decreased at 6.67 and 10 mg/L ß-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 ß-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 ß-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 ß-ionone on the PS II of M. aeruginosa NIES-843.

[Correlation analysis among characters of gas vesicle in Microcystis strains].

In order to explore the roles of gvpA copies and repeated sequences of gvpC, three Microcystis strains including M. aeruginosa FACHB910, M. aeruginosa FACHB930 and M. wesenbergii FACHB929 were used in this research. The length and diameter of gas vesicle, relative gas vesicle volume, appear pressure values, critical pressure values, cell turgor values were measured, and the correlations among these characters, gvpA copies, and repeated sequences of gvpC were analyzed. The results indicate that there are a significant positive linear correlation between gvpA copies and relative gas vesicle volume (r = 0.999); gvpA copies are negatively correlated with diameter of gas vesicle (r = -0.861). However, repeated sequences of gvpC have a significant positive correlation with diameter of gas vesicle (r = 0.911), and have significant negative correlations with relative gas vesicle volume, appear pressure values and critical pressure values, with the correlation coefficient of -0.851, - 0.999, - 0.928 respectively. So we presume that gvpA copies are probably the primary impact factor for relative gas vesicle volume. The diameter of gas vesicle is not only regulated by repeated sequences of gvpC, but also regulated by gvpA copies.

Immunogold localisation of microcystins in cryosectioned cells of Microcystis.

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.

The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length.

In cyanobacteria the protein on the outside of the gas vesicle, GvpC, is characterised by the presence of a 33 amino acid residue repeat (33RR), which in some genera is highly conserved. The number of 33RRs correlates with the diameter of the gas vesicle and inversely with its strength. Gas vesicles isolated from Microcystis aeruginosa strain PCC 7806 were found to be wider and have a lower critical collapse pressure than those from Microcystis sp. strain BC 8401. The entire gas-vesicle gene cluster of the latter strain was sequenced and compared with the published sequence of the former: the sequences of nine of the ten gvp genes differed by only 1-5% between the two strains; the only substantial difference was in gvpC which in strain BC 8401 lacked a 99-nucleotide section encoding a 33RR. This observation further narrows the correlation of gas vesicle width to the number of 33RRs and suggests how Microcystis strains might be used in experimental manipulation of gas vesicle width and strength.

Mobile DNA elements in the gas vesicle gene cluster of the planktonic cyanobacteria Microcystis aeruginosa.

Insertion sequences (IS) have been characterized in Microcystis aeruginosa gas vesicle-deficient mutants. ISMae4, a homolog of the cyanobacterial IS702, belongs to the IS5 family, subgroup ISL2. ISMae2 and ISMae3 display typical IS features and express a transposase of the IS4 and IS1 family, respectively. ISMae1 exhibits a more complex genetic structure and harbours a degenerated transposase gene distantly related to IS1 elements. Hybridizations with IS-specific DNA probes suggest that transposition of ISMae2 and ISMae3 occurred by a replicative-type mechanism. To our knowledge this is the first report showing that IS1 elements can be mobile in cyanobacteria.

Intracellular phosphorus metabolism of Microcystis aeruginosa under various redox potential in darkness.

Phosphorus metabolism of Microcystis aeruginosa was studied under gradient redox potential from 252 mV to -70 mV in darkness. The release of phosphorus occurred in all the treatments, and this process was accelerated in darkness when the redox potential was lowered. Low redox potential in darkness stimulated the accumulation of polyphosphate (PolyP) and the degradation of polyglucose. The synthesis of PolyP delayed the decrease of intracellular orthophosphate. The death of M. aeruginosa was slowered when the redox potential was low in darkness. The accumulation of PolyP under low redox potential in the dark was very important to M. aeruginosa for endurance through the unfavorable growth conditions for maintaining phosphorus concentration, energy storage, and other physiological functions. The ability to accumulate PolyP in the dark and negative redox potential may be of considerable advantage in the low-light, organically rich, and low-redox habitats.