Phylogenomics Uncovers Evolutionary Trajectory of Nitrogen Fixation in Cyanobacteria.

Biological nitrogen fixation (BNF) by cyanobacteria is of significant importance for the Earth's biogeochemical nitrogen cycle but is restricted to a few genera that do not form monophyletic group. To explore the evolutionary trajectory of BNF and investigate the driving forces of its evolution, we analyze 650 cyanobacterial genomes and compile the database of diazotrophic cyanobacteria based on the presence of nitrogen fixation gene clusters (NFGCs). We report that 266 of 650 examined genomes are NFGC-carrying members, and these potentially diazotrophic cyanobacteria are unevenly distributed across the phylogeny of Cyanobacteria, that multiple independent losses shaped the scattered distribution. Among the diazotrophic cyanobacteria, two types of NFGC exist, with one being ancestral and abundant, which have descended from diazotrophic ancestors, and the other being anaerobe-like and sparse, possibly being acquired from anaerobic microbes through horizontal gene transfer. Interestingly, we illustrate that the origin of BNF in Cyanobacteria coincide with two major evolutionary events. One is the origin of multicellularity of cyanobacteria, and the other is concurrent genetic innovations with massive gene gains and expansions, implicating their key roles in triggering the evolutionary transition from nondiazotrophic to diazotrophic cyanobacteria. Additionally, we reveal that genes involved in accelerating respiratory electron transport (coxABC), anoxygenic photosynthetic electron transport (sqr), as well as anaerobic metabolisms (pfor, hemN, nrdG, adhE) are enriched in diazotrophic cyanobacteria, representing adaptive genetic signatures that underpin the diazotrophic lifestyle. Collectively, our study suggests that multicellularity, together with concurrent genetic adaptations contribute to the evolution of diazotrophic cyanobacteria.

The facilitating role of phycospheric heterotrophic bacteria in cyanobacterial phosphonate availability and Microcystis bloom maintenance.

BACKGROUND: Phosphonates are the main components in the global phosphorus redox cycle. Little is known about phosphonate metabolism in freshwater ecosystems, although rapid consumption of phosphonates has been observed frequently. Cyanobacteria are often the dominant primary producers in freshwaters; yet, only a few strains of cyanobacteria encode phosphonate-degrading (C-P lyase) gene clusters. The phycosphere is defined as the microenvironment in which extensive phytoplankton and heterotrophic bacteria interactions occur. It has been demonstrated that phytoplankton may recruit phycospheric bacteria based on their own needs. Therefore, the establishment of a phycospheric community rich in phosphonate-degrading-bacteria likely facilitates cyanobacterial proliferation, especially in waters with scarce phosphorus. We characterized the distribution of heterotrophic phosphonate-degrading bacteria in field Microcystis bloom samples and in laboratory cyanobacteria "phycospheres" by qPCR and metagenomic analyses. The role of phosphonate-degrading phycospheric bacteria in cyanobacterial proliferation was determined through coculturing of heterotrophic bacteria with an axenic Microcystis aeruginosa strain and by metatranscriptomic analysis using field Microcystis aggregate samples. RESULTS: Abundant bacteria that carry C-P lyase clusters were identified in plankton samples from freshwater Lakes Dianchi and Taihu during Microcystis bloom periods. Metagenomic analysis of 162 non-axenic laboratory strains of cyanobacteria (consortia cultures containing heterotrophic bacteria) showed that 20% (128/647) of high-quality bins from eighty of these consortia encode intact C-P lyase clusters, with an abundance ranging up to nearly 13%. Phycospheric bacterial phosphonate catabolism genes were expressed continually across bloom seasons, as demonstrated through metatranscriptomic analysis using sixteen field Microcystis aggregate samples. Coculturing experiments revealed that although Microcystis cultures did not catabolize methylphosphonate when axenic, they demonstrated sustained growth when cocultured with phosphonate-utilizing phycospheric bacteria in medium containing methylphosphonate as the sole source of phosphorus. CONCLUSIONS: The recruitment of heterotrophic phosphonate-degrading phycospheric bacteria by cyanobacteria is a hedge against phosphorus scarcity by facilitating phosphonate availability. Cyanobacterial consortia are likely primary contributors to aquatic phosphonate mineralization, thereby facilitating sustained cyanobacterial growth, and even bloom maintenance, in phosphate-deficient waters. Video Abstract.

The widespread capability of methylphosphonate utilization in filamentous cyanobacteria and its ecological significance.

Aquatic ecosystems comprise almost half of total global methane emissions. Recent evidence indicates that a few strains of cyanobacteria, the predominant primary producers in bodies of water, can produce methane under oxic conditions with methylphosphonate serving as substrate. In this work, we have screened the published 2 568 cyanobacterial genomes for genetic elements encoding phosphonate-metabolizing enzymes. We show that phosphonate degradation (phn) gene clusters are widely distributed in filamentous cyanobacteria, including several bloom-forming genera. Algal growth experiments revealed that methylphosphonate is an alternative phosphorous source for four of five tested strains carrying phn clusters, and can sustain cellular metabolic homeostasis of strains under phosphorus stress. Liberation of methane by cyanobacteria in the presence of methylphosphonate occurred mostly during the light period of a 12 h/12 h diurnal cycle and was suppressed in the presence of orthophosphate, features that are consistent with observations in natural aquatic systems under oxic conditions. The results presented here demonstrate a genetic basis for ubiquitous methane emission via cyanobacterial methylphosphonate mineralization, while contributing to the phosphorus redox cycle.

Comparative genomics reveals insights into cyanobacterial evolution and habitat adaptation.

Cyanobacteria are photosynthetic prokaryotes that inhabit diverse aquatic and terrestrial environments. However, the evolutionary mechanisms involved in the cyanobacterial habitat adaptation remain poorly understood. Here, based on phylogenetic and comparative genomic analyses of 650 cyanobacterial genomes, we investigated the genetic basis of cyanobacterial habitat adaptation (marine, freshwater, and terrestrial). We show: (1) the expansion of gene families is a common strategy whereby terrestrial cyanobacteria cope with fluctuating environments, whereas the genomes of many marine strains have undergone contraction to adapt to nutrient-poor conditions. (2) Hundreds of genes are strongly associated with specific habitats. Genes that are differentially abundant in genomes of marine, freshwater, and terrestrial cyanobacteria were found to be involved in light sensing and absorption, chemotaxis, nutrient transporters, responses to osmotic stress, etc., indicating the importance of these genes in the survival and adaptation of organisms in specific habitats. (3) A substantial fraction of genes that facilitate the adaptation of Cyanobacteria to specific habitats are contributed by horizontal gene transfer, and such genetic exchanges are more frequent in terrestrial cyanobacteria. Collectively, our results further our understandings of the adaptations of Cyanobacteria to different environments, highlighting the importance of ecological constraints imposed by the environment in shaping the evolution of Cyanobacteria.

Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120.

Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P(i)) during P(i) starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P(i) starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP(A), coded by all2843, is characterized as a metalloenzyme containing Mg2+ and Zn2+ with molar ratio of 1 : 2. Site-directed mutagenesis analysis indicated that, though the active center of AP(A) is highly conserved in comparison with EAP, differences do exist between AP(A) and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP(A) is a monomeric protein and inactivated rapidly at 50 degrees C. These results suggest that AP(A) is the first monomeric heat-labile classical PhoA found in cyanobacteria.

Prediction of different stages of rectal cancer: Texture analysis based on diffusion-weighted images and apparent diffusion coefficient maps.

BACKGROUND: It is evident that an accurate evaluation of T and N stage rectal cancer is essential for treatment planning. It has not been extensively investigated whether texture features derived from diffusion-weighted imaging (DWI) images and apparent diffusion coefficient (ADC) maps are associated with the extent of local invasion (pathological stage T1-2 vs T3-4) and nodal involvement (pathological stage N0 vs N1-2) in rectal cancer. AIM: To predict different stages of rectal cancer using texture analysis based on DWI images and ADC maps. METHODS: One hundred and fifteen patients with pathologically proven rectal cancer, who underwent preoperative magnetic resonance imaging, including DWI, were enrolled, retrospectively. The ADC measurements (ADCmean, ADCmin, ADCmax) as well as texture features, including the gray level co-occurrence matrix parameters, the gray level run-length matrix parameters and wavelet parameters were calculated based on DWI (b = 0 and b = 1000) images and the ADC maps. Independent sample t-tests or Mann-Whitney U tests were used for statistical analysis. Multivariate logistic regression analysis was conducted to establish the models. The predictive performance was validated by receiver operating characteristic curve analysis. . RESULTS: Dissimilarity, sum average, information correlation and run-length nonuniformity from DWI b =0 images, gray level nonuniformity, run percentage and run-length nonuniformity from DWI b =1000 images, and dissimilarity and run percentage from ADC maps were found to be independent predictors of local invasion (stage T3-4). The area under the operating characteristic curve of the model reached 0.793 with a sensitivity of 78.57% and a specificity of 74.19%. Sum average, gray level nonuniformity and the horizontal components of symlet transform (SymletH) from DWI b =0 images, sum average, information correlation, long run low gray level emphasis and SymletH from DWI b =1000 images, and ADCmax, ADCmean and information correlation from ADC maps were identified as independent predictors of nodal involvement. The area under the operating characteristic curve of the model reached 0.802 with a sensitivity of 80.77% and a specificity of 68.25%. CONCLUSION: Texture features extracted from DWI images and ADC maps are useful clues for predicting pathological T and N stages in rectal cancer.

Response of microcystis to copper stress: do phenotypes of microcystis make a difference in stress tolerance?

To elucidate the role of phenotype in stress-tolerant bloom-forming cyanobacterium Microcystis, two phenotypes of M. aeruginosa - unicellular and colonial strains were selected to investigate how they responded to copper stress. Flow cytometry (FCM) examination indicated that the percents of viable cells in unicellular and colonial Microcystis were 1.92-2.83% and 72.3-97.51%, respectively, under 0.25 mgl(-1) copper sulfate treatment for 24h. Upon exposure to 0.25 mgl(-1) copper sulfate, the activities of antioxidative enzyme, such as superoxide dismutase (SOD) and catalase (CAT), were significantly increased in colonial Microcystis compared to unicellular Microcystis. Meanwhile, the values of the photosynthetic parameters (F(v)/F(m), ETR(max), and oxygen evolution rate) decreased more rapidly in unicellular Microcystis than in colonial Microcystis. The results indicate that colonial Microcystis has a higher endurance to copper than unicellular Microcystis. This suggests that the efficient treatment concentration of copper sulfate as algaecides will be dependent on the phenotypes of Microcystis.

[Acute toxicity of microcystin-LR in BALB/c mice].

OBJECTIVE: To investigate acute toxicity of microcystin-LR in BALB/c mice. METHODS: BALB/c mice were subjected to intraperitoneal injection of microcystin-LR at the half lethal dose (LD50) and 1/2 LD50, and the organ weight indices and various biochemical indices were determined 30 min and 4 h after the treatment, respectively. RESULTS: Significant increase in liver or kidney weight index occurred in the treated mice, indicating hemorrhagic conditions of the two organs. A slight increase in serum levels of aspartate amino transferase and alanine aminotransferase was observed along with an obvious decrease in serum urea levels, suggesting liver damage. The increase of serum uric acid also indicated the presence of kidney damage. CONCLUSION: The kidney may be another target of microcystins besides the liver.

An ELISA-like time-resolved fluorescence immunoassay for microcystin detection.

BACKGROUND: A time-resolved fluorescence immunoassay (TRFIA), based on anti-microcystin-LR (MCLR) monoclonal antibodies (MAbs) and europium-labeled antimouse IgG conjugate, was first developed for microcystin detection. METHODS: Anti-MCLR MAbs were prepared by a standard method, and the attained MAbs showed a good cross reactivity with MCLR, MCRR and MCYR. The TRFIA was performed in an indirect competitive mode. The detection method of TRFIA was compared with indirect competitive enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). RESULTS: The TRFIA exhibited a typical sigmoidal response for MCLR at concentrations of 0.005-50 ng/ml, with a wide quantitative range between 0.01 and 10 ng/ml, indicating the broadest detective range and the most sensitive of all the methods for microcystins (MCs) detection. Additionally, the TRFIA maintained good reliability through its quantitative range, as evidenced by low coefficients of variation (1.6-12.2%). The toxin data of algal samples assayed from TRFIA were in the same range as those with ELISA and HPLC, implying that the method was reliable and practical for the detection of MCs. CONCLUSIONS: The TRFIA may offer a valuable alternative or a substitute for conventional ELISA for microcystin detection.

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity.

It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of glutathione [correction of gluathione], an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity.

[Lipid peroxidation in microalgae cells under simulated microgravity].

Objective. To provide direct evidences for effects of microgravity on structure and function of plasma membrane. Method. Malondialdehyde (MDA) content was examined on the basis of quantitative reaction of both MDA and thiobarbituric acid (TBA), and electrolyte leaking was determined with conductometer model DDS-11A. Result. Experiments showed that under simulated microgravity, lipid peroxidation and the content of MDA increased. Meanwhile, the membrane permeability increased in cells of two microalgae: Anabaena sp PCC7120 and Synechococcus 7942. Conclusion. Our results suggest that there is some commonness between microgravity stress and certain other environmental stresses. And cellular membrane might be the site of perception of gravity in unicells without special gravity sensitive structure, such as alga cells.

Cyanobacterial fossils from 252†Ma old microbialites and their environmental significance.

The end-Permian mass extinction was followed by the formation of an enigmatic rock layer with a distinctive macroscopic spotted or dendroid fabric. This deposit has been interpreted as microbial reef rock, digitate dendrolite, digital thrombolite, dendritic thrombolite, or bacterial deposits. Agreement has been reached in considering them as microbialites, but not in their formation. This study has revealed that the spotted and dendroid microbialites were composed of numerous fossil casts formed by the planktic cyanobacterium, Microcystis, a coccoid genus that at the present-day commonly forms blooms in modern lakes, rivers, and reservoirs. The abundance of the fossils and the diagenesis they experienced has determined the macroscopic fabric: where they abundant, the rock appears as dendroid, otherwise, it appears as spotted. The ancient Microcystis bloom might produce toxin to kill other metazoans, and be responsible for the oceanic anoxia that has puzzled so many researchers for so many years.

[Dianchi Lake macroinvertebrate community succession trends and retrogressive analysis].

Historical records and data from yield surveys conducted in 2009 and 2010 were used to investigate macroinvertebrate community succession trends in Dianchi Lake. Species richness has declined from 57 in the 1980s to 32 in 2010, representing a species loss of 44%. Among the major benthic groups, the highest rate of loss was recorded for mollusks (75%) and aquatic insects (39%). Surveys in 2009 and 2010 across the lake revealed that the total density was 1776 ind/m2, comprising oligochaetes (1706 ind/m2) and chironomids (68 ind/m2). Over a nearly twenty-year span (1992-2010), the density and biomass of oligochaetes first increased sharply (1992-2002) and then declined gradually (2002-2010). Further, chironomids have decreased gradually while the proportion of abundant species has increased. Limnodrilus hoffmeisteri became the sole dominant species with an average relative abundance of 74.1%. Cosmopolitan species, such as Einfeldia sp., disappeared across the lake; instead, tolerant species such as Chironomus plumosus, Ch. attenuatus and Tanypus chinensis became the common. Mollusk community structure has become simpler and many native species have gone extinct. Species of concern include Margarya melanioides, M. mondi, M. mansugi and Cipangopaludina dianchiensis, all rated as critically endangered by the IUCN. We found that the Shannon-Wiener index declined in Dianchi Lake, particularly in Caohai Lake, from 2.70 in the 1950s to 0.30 in 2009 and 2010. Species richness and biodiversity was significantly negative correlated with total phosphorus and total nitrogen. Factors responsible for the benthic community retrogression described here include habitat destruction, lowering of water quality, outbreaks of blue-green algae, extinction of submerged plants and lack of germplasm resources.

[Physiological character and the ratio of the sinking Microcystis in Lake Dianchi].

Aimed to explore the relationship between Microcystis cells sinking and water bloom development, the physiological status of the sinking cells was characterized by comparing with the floating cells. The sinking ratio and the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) staining were also employed in surface and bottom samples to substantiate the character. The dynamics of the sinking ratio and the chlorophyll a concentration were also compared. The field Microcystis cells had relative stable positions in the sample container after 2 hours settlement in the lab. Statistic analysis suggested that the floating and the sinking colonies did not differ significantly on the indicators maximum quantum yield of PS II and membrane integrity (p > 0. 05), but did significantly on the electron transfer rate and nicotinamide adenine dinucleotide (phosphate), reduced disodium salt [ NAD (P) H] dependent oxidoreductase and dehydrogenase activities (p < 0.01). Furthermore, comparison between bottom samples and surface samples suggested the bottom had higher ratio of sinking and MTT negative cells (p < 0.01). These results implied that the sinking cells were injured and had lower metabolic activity. During the time from October 2007 to May 2008, the sinking ratio reached climax about 40% in winter, and a negative correlation between the sinking ratio and chlorophyll a concentration was observed (p < 0.05). Sinking played an important role in the decline of Microcystis bloom. A research on the sinking ratio will be helpful for the understanding of bloom development tendency. The characterization on the physiology status of sinking cells constructed basement for the introduction of sinking ratio to the prediction of water bloom development.

[Recruitment ability of Microcystis aeruginosa under low light-low temperature combination].

The responses of growth and PS II activities in Microcystis aeruginosa (FACHB 905) have been studied under a condition of low light-temperature combination use orthogonal experiment method. The contents and proportions of chlorophyll and carotenoid were determined by colorimetry, the PS II activities were assayed with a Water-PAM, and also, the photosynthesis recovery of M. aeruginosa was verified via reculture under a normal condition. The results showed that recruitment of M. aeruginosa should not be triggered since it could hardly grow under the temperature of 9 degrees C. Under 12 degrees C, the growth was greatly affected by the light intensity. 12 degrees C & 100 lx combination was considered to be the threshold value to induce recruitment of Microcystis due to the physiological responses in growth and photosynthetic system. The growth of alga was obviously inhibited in all samples. However, the biomass under 15 degrees C & 100 lx combination was the largest, which reached about 0.88 mg/L, and it was about 2-17 times compared to the other samples, respectively. We also found FACHB 905 could persist longer under low light intensity (100 lx) than a relative higher intensity (500 lx) under 15 degrees C, since the chlorophyll content, electron transfer rate and yield were relative higher in combination. Reculture of M. aeruginosa was conducted after a 20 d study, samples under the temperatures of 9 degrees C & 12 degrees C recovered soon in growth characters and PS II activities during 5 days. Meanwhile, all the samples of FACHB 905 reached a rather stable growing status, with a fluorescence quantum yield about 0.55-0.6, like other normal cultured samples finally. The present results should be important to determine the tolerance threshold and even to reveal the probable mechanisms in overwintering and recruitment of M. aeruginosa from lake sediments.

[One step time-resolved fluorescence immunoassay for direct estimation of microcystins].

A direct-competitive time-resolved fluorescence immunoassay (TRFIA) for microcystin detection was established, which was based on europium labeled MCLR-BSA conjugant and microtiter plates coated with anti-mouse IgG. The optimal dilution of europium labeled MCLR-BSA conjugant is 1/50 and most appropriate titration of anti-microcystin-LR (MCLR) monoclonal antibodies is 100 ng/mL. The standard curve under the optimal conditions shows that the quantitative range is from approximately 0.05 ng/mL to 10 ng/mL. The detection limit of the method is 0.02 ng/mL for MCLR, recoveries for microcystin (> 94%) in quantitative range is satisfactory.

[Microcystin-LR induces apoptosis in L-02 cell line].

OBJECTIVE: To investigate the toxicological mechanism of microcystin-LR (MCLR) on L-02 cells. METHODS: L-02 cells was treated with MCLR at different concentrations and the subsequent changes such as cell proliferation (MTT assay), morphology, lactate dehydrogenase (LDH) leakage, apoptosis rate and apoptosis-related gene expression were examined. RESULTS: MTT assay showed that MCLR mildly inhibited the cell growth within the initial 24 h of treatment but enhanced the cell viability after that till 60 h in a time- and dose-dependent manner. LDH leakage underwent no marked changes in response to 48-hour MCLR treatment but increased upon prolonged treatment for 60 h, indicating the presence of oxidative damage. After a 48-h treatment with MCLR at 50 microg/ml, obvious apoptosis of L-02 cells occurred as manifested by cell rounding, detachment from the substrate, cell shrinkage and membrane blebbing. The apoptosis rates were rather low (between 22% and 29%) after treatment with MCLR at different concentrations for 36 h, and increased to as much as 80% after a 60-h treatment with 50 microg/ml MCLR. The expressions of p53 and bcl-2 increased in the cells after treatment with high-concentration MCLR, suggesting that MCLR up-regulated the expression levels of the two proteins. CONCLUSION: MCLR can induce apoptosis and up-regulate p53 and bcl-2 expressions in human normal liver cell line L-02.