Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

UV-acclimation responses in natural populations of cyanobacteria (Calothrix sp.)

Phenotypic acclimation to changing conditions is typically thought to be beneficial to organisms in the environment. UV radiation is an important parameter affecting photosynthetic organisms in natural environments. We measured the response of photosynthetic carbon fixation in populations of cyanobacteria inhabiting a hot spring following acclimation to different UV treatments. These two very closely related populations of cyanobacteria, differing in their content of the extracellular UV-screening pigment scytonemin, were acclimated in situ under natural solar irradiance modified by filters that excluded both UVA/B, only UVB or transmitted both UVA/B. Cells from each preacclimation treatment were subsequently assayed for photosynthetic performance under all UV conditions (incubation treatment) giving a two-factor experimental design for each population. No acclimation filter treatment effects were observed even after two months under different acclimation treatments. This suggests that UV photoacclimation does not occur in either of these populations, regardless of the presence of scytonemin. By contrast, cells showed significant UV-inhibition during 1 h incubations under full sun. The population with high levels of scytonemin usually had lower rates of photosynthetic carbon fixation than the scytonemin-lacking population. However, the degree of UV inhibition, especially UVA inhibition, was higher for the cells without scytonemin pigment. These results suggest that closely related natural cyanobacterial populations respond differently to natural irradiance conditions and may be adopting different strategies of UV tolerance.     

Optimum conditions for growth of cyanobacteria on solid media

The colony forming ability of single cells or very short filaments of 7 strains of cyanobacteria was tested on media solidified by agar or by agar substitutes (Gel Gro or Gel Rite). In addition, the effect of various methods for preparation of agar media on colony forming ability was measured. High efficiency colony formation for most of the strains required that the agar be autoclaved separately from the salts in the medium. The addition of thiosulfate, but not buffer, significantly increased the plating efficiency of most strains.     

Environmental conditions in high mountain lakes containing toxic benthic cyanobacteria

In glacial lakes on an alpine pasture in Switzerland, benthic cyanobacteria produced microcystin, a cyclic hepatotoxic heptapeptide. The cyanobacteria formed dense mats on sediments and submerged stones. The mats consisted mainly of Oscillatoria limosa, Phormidium konstantinosum (= Oscillatoria tenuis) and Tychonema granulatum (= Oscillatoria granulata). In order to characterize the ecological conditions of these cyanobacteria, nutrient concentrations were determined, and an automatic data acquisition station was installed in one of the lakes. It continuously recorded air temperature, global irradiance, precipitation, atmospheric pressure, wind speed and direction; as well as temperature, pH, oxygen content and conductivity of the lake water. The nutrient situation in the lakes was mainly influenced by the erosion of the gneissic catchment by glacial meltwater and by precipitation. In the glacial lakes, the concentrations of calcium, iron, magnesium, sodium and sulphate increased throughout the summer season. Conductivity values of 4–110 μS cm^-1 represent generally low nutrient concentrations. Nevertheless, iron concentrations of up to 20 μM occurred. Biomass, expressed as protein concentration, as well as the microcystin content of the cyanobacterial mats varied within one season and between different years (1994 and 1995). In one cyanobacterial mat community, biomass and microcystin concentrations were highest at the same time, in an other one the microcystin content was maximal three weeks after the highest biomass concentration was reached. Our observations suggest that biomass and toxin production in the mats were strongly influenced by mechanical stress, temporary desiccation and high irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bioactive natural products from marine cyanobacteria for drug discovery

The prokaryotic marine cyanobacteria continue to be an important source of structurally bioactive secondary metabolites. A majority of these molecules are nitrogen-containing compounds biosynthesized by large multimodular nonribosomal polypeptide (NRP) or mixed polyketide-NRP enzymatic systems. A total of 128 marine cyanobacterial alkaloids, published in the literature between January 2001 and December 2006, are presented in this review with emphasis on their biosynthesis and biological activities. In addition, a number of highly cytotoxic compounds such as hectochlorin, lyngbyabellins, apratoxins, and aurilides have been identified as potential lead compounds for the development of anticancer agents. A brief coverage on the distribution of natural product biosynthetic genes as well as the mechanisms of tailoring enzymes involved in the biosynthesis of cyanobacterial compounds will also be given.     

Circadian programming in cyanobacteria.

Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle. This biological clock orchestrates global regulation of gene expression. Competition experiments demonstrate that fitness is enhanced when the circadian period is consonant with the period of the environmental cycle. Mutational analyses have identified three clock genes in the organism, one of which is related to DNA recombinases and helicases. We propose a new model for the core 'clockwork' that implicates rhythmic changes in the status of the chromosome that underly the rhythms of gene expression.     

Proteomic analyses of the response of cyanobacteria to different stress conditions

Cyanobacteria are significant contributors to global photosynthetic productivity, thus making it relevant to study how the different environmental stresses can alter their physiological activities. Here, we review the current research work on the response of cyanobacteria to different kinds of stress, mainly focusing on their response to metal stress as studied by using the modern proteomic tools. We also report a proteomic analysis of plastocyanin and cytochrome c₆ deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803 grown under copper or iron deprivation, as compared to wild-type cells, so as to get a further understanding of the metal homeostasis in cyanobacteria and their response to changing environmental conditions.     

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reversed-phase liquid chromatographic-mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions

Reversed-phase HPLC coupled to the atmospheric pressure ionization-electrospray ionization (API-ESI) MS was used for microcystin-LR detection and quantitation in samples of dried Microcystis aeruginosa cells. An alkaline linear gradient (20 mmol/l ammonium hydroxide-acetonitrile, pH 9.7) was used for elution of the toxic peptides. Limit of detection was 1 microg/ml (20 ng per injection) in the scan mode of MS and 0.1 microg/ml (2 ng per injection) in the case of selective ion monitoring.     

Hydrogen production by cyanobacteria in an automated outdoor photobioreactor under aerobic conditions

The possibility of hydrogen production by a hydrogenase impaired mutant strain of Anabaena variabilis in outdoor culture was studied. A computer-controlled rooftop (outdoor) tubular photobioreactor (4.35 L) was assembled. H(2) production rates by A. variabilis PK84 grown in CO(2) + air in the photobioreactor were measured together with other parameters such as temperature, irradiance, pH, dry biomass weight, and pO(2), and Chl a concentrations during summer months of 1998 and 1999. Efficiencies of light energy bioconversion to H(2) energy and energy accumulated in biomass were calculated. The influence of irradiance, temperature, and mode of cultivation on H(2) production and efficiency of light energy bioconversion were evaluated. The culture produced up to 1.1 L H(2) day(-1) PhBR(-1). The efficiency of light energy to H(2) energy bioconversion on some days was 0.094%. However, the conditions for maximum H(2) photoproduction and for maximum efficiency of light energy to H(2) energy bioconversion were not the same. A. variabilis PK84 could produce hydrogen for prolonged periods (up to 40 days) without injection of fresh inoculum. During this period photobioreactor produced 24.5 L of H(2). Possibilities for increasing the efficiency of light energy conversion are discussed.     

Genome mining for natural product biosynthetic gene clusters in the Subsection V cyanobacteria

Background

Cyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons.

Results

A putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared.

Conclusions

Genome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1855-z) contains supplementary material, which is available to authorized users.     

Ecology of Gordian knots in natural conditions

The ecology of “Gordian knots” – aggregates of entwined reproductive individuals of nematomorphs, or hair worms – remains poorly understood. We studied the ecology of Gordian knots in a wild population of the hair worm Paragordius tricuspidatus sampled from a stream near Montpellier, France, in 2005 and 2006. At our study site, knots were composed entirely of males. The number of adults per knot varied throughout the summer, most likely due to the seasonal decrease in stream water level and the appearance of females later in the season. The average length of individual worms in each knot did not change significantly over time, but it became significantly more variable. We also showed that knots within these streams were more likely to be attached to floating rather than immobile substrates. Because the water levels in our sampling stream decrease as the season progresses, males in knots attached to floating substrates could be in a better position to cope with decreasing water levels, as they always remain immersed. An important avenue of future study would be to explore the effects of varying water level and the presence of females on the size and maintenance of knots in laboratory conditions.     

Survival of enteric viruses under natural conditions in a subarctic river.

The survival of enteric viruses was studied in the vicinity of Fairbanks, Alaska at selected stations along a 317-km section of the Tanana River. This section was located downstream from all known domestic wastewater sources and was effectively sealed by a total ice cover. The mean flow time through the region was 7.1 days, during which initial viral population showed a relative survival rate of 34%. The tracing of native viruses at such great distances in the complete absence of other point and nonpoint viral sources has not been previously reported. Of the two methods of virus concentration used, viral recoveries from the disk adsorption virus elution procedure were far greater than those achieved with the Aquella system employed at that time. The fact the ratio of enteric viruses to fecal indicator bacteria was not constant clearly inferred that these bacteria were not an effectual measure of virus concentration. The persistence of fecal coliforms and fecal streptococci, however, attested to the microbiological health risk involved.     

Survival of enteric viruses under natural conditions in a subarctic river.

The survival of enteric viruses was studied in the vicinity of Fairbanks, Alaska at selected stations along a 317-km section of the Tanana River. This section was located downstream from all known domestic wastewater sources and was effectively sealed by a total ice cover. The mean flow time through the region was 7.1 days, during which initial viral population showed a relative survival rate of 34%. The tracing of native viruses at such great distances in the complete absence of other point and nonpoint viral sources has not been previously reported. Of the two methods of virus concentration used, viral recoveries from the disk adsorption virus elution procedure were far greater than those achieved with the Aquella system employed at that time. The fact the ratio of enteric viruses to fecal indicator bacteria was not constant clearly inferred that these bacteria were not an effectual measure of virus concentration. The persistence of fecal coliforms and fecal streptococci, however, attested to the microbiological health risk involved.     

Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation

Microalgae are regarded as a potential biomass source for biofuel purposes. With regard to bioethanol production, microalgae seem to overcome traditional substrate drawbacks. Enzymatic activities are responsible for carbon allocation and hence for carbohydrate profiles. Enzyme activities may be manipulated by metabolic engineering; however, this goal may also be achieved by controlling environmental conditions of the culture system. We outline the key-enzymes as well as the main operational conditions applied to microalgae growth (inorganic nutrient supplementation, irradiance and temperature) that affect carbohydrate synthesis on microalgae and cyanobacteria. Normally, harsh conditions are needed for such a goal and thus, arrested microalgae growth may occur. Potential strategies to avoid arrested growth, while enhancing carbohydrate accumulation, were also pointed out in this review.