Increased algicidal activity of Aeromonas veronii in response to Microcystis aeruginosa: interspecies crosstalk and secondary metabolites synergism.

Toxic Microcystis spp. blooms constitute a serious threat to water quality worldwide. Aeromonas veronii was isolated from Microcystis sp. colonies collected in Lake Kinneret. Spent Aeromonas media inhibits the growth of Microcystis aeruginosa MGK isolated from Lake Kinneret. The inhibition was much stronger when Aeromonas growth medium contained spent media from MGK suggesting that Aeromonas recognized its presence and produced secondary metabolites that inhibit Microcystis growth. Fractionations of the crude extract and analyses of the active fractions identified several secondary metabolites including lumichrome in Aeromonas media. Application of lumichrome at concentrations as low as 4 nM severely inhibited Microcystis growth. Inactivation of aviH in the lumichrome biosynthetic pathway altered the lumichrome level in Aeromonas and the extent of MGK growth inhibition. Conversely, the initial lag in Aeromonas growth was significantly longer when provided with Microcystis spent media but Aeromonas was able to resume normal growth. The longer was pre-exposure to Microcystis spent media the shorter was the lag phase in Aeromonas growth indicating the presence of, and acclimation to, secondary MGK metabolite(s) the nature of which was not revealed. Our study may help to control toxic Microcystis blooms taking advantage of chemical languages used in the interspecies communication.

Does 2-phosphoglycolate serve as an internal signal molecule of inorganic carbon deprivation in the cyanobacterium Synechocystis sp. PCC 6803?

Cyanobacteria possess CO2 -concentrating mechanisms (CCM) that functionally compensate for the poor affinity of their ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to CO2 . It was proposed that 2-phosphoglycolate (2PG), produced by the oxygenase activity of Rubisco and metabolized via photorespiratory routes, serves as a signal molecule for the induction of CCM-related genes under limiting CO2 level (LC) conditions. However, in vivo evidence is still missing. Since 2PG does not permeate the cells, we manipulated its internal concentration. Four putative phosphoglycolate phosphatases (PGPases) encoding genes (slr0458, sll1349, slr0586 and slr1762) were identified in the cyanobacterium Synechocystis PCC 6803. Expression of slr0458 in Escherichia coli led to a significant rise in PGPase activity. A Synechocystis mutant overexpressing (OE) slr0458 was constructed. Compared with the wild type (WT), the mutant grew slower under limiting CO2 concentration and the intracellular 2PG level was considerably smaller than in the wild type, the transcript abundance of LC-induced genes including cmpA, sbtA and ndhF3 was reduced, and the OE cells acclimated slower to LC - indicated by the delayed rise in the apparent photosynthetic affinity to inorganic carbon. Data obtained here implicated 2PG in the acclimation of this cyanobacterium to LC but also indicated that other, yet to be identified components, are involved.

Cross-talk between photomixotrophic growth and CO(2) -concentrating mechanism in Synechocystis sp. strain PCC 6803.

Simultaneous catabolic and anabolic glucose metabolism occurs in the same compartment during photomixotrophic growth of the model cyanobacterium Synechocystis sp. PCC 6803. The presence of glucose is stressful to the cells; it is reflected in the high frequency of suppression mutations in glucose-sensitive mutants. We show that glucose affects many cellular processes. It stimulates respiration and the rate of photosynthesis and quantum yield in low- but not high-CO(2) -grown cells. Fluorescence and thermoluminescence parameters of photosystem II are also affected but the results did not lend support to sustained glucose driven over reduction in the light. Glucose-sensitive mutants such as ΔpmgA (impaired in photomixotrophic growth) and Δhik31 (lacking histidine kinase 31) are far more susceptible under high than low air level of CO(2) . A glycine to tryptophan mutation in position 354 in NdhF3, involved in the high-affinity CO(2) uptake, rescued ΔpmgA. A rise in the apparent photosynthetic affinity to external inorganic carbon is observed in high-CO(2) -grown wild-type cells after the addition of glucose, but not in mutant ΔpmgA. This is attributed to upregulation of certain low-CO(2) -induced genes, involved in inorganic carbon uptake, in the wild type but not in ΔpmgA. These data uncovered a new level of interaction between CO(2) fixation (and the CO(2) -concentrating mechanism) and photomixotrophic growth in cyanobacteria.

A cyanobacterial AbrB-like protein affects the apparent photosynthetic affinity for CO2 by modulating low-CO2-induced gene expression.

In Synechocystis sp. strain PCC 6803, over 450 genes are upregulated following transfer of the cells from a high (1-5% CO(2) in air, HC) to a low level of CO(2) (as in air or lower, LC). This includes sbtA, ndhF3 and cmpA involved in inorganic carbon (Ci) uptake. Earlier studies implicated NdhR in the regulation of LC-induced genes but there are indications that additional components are involved. Following extraction of proteins from cells grown under HC and (NH4)(2)SO(4) fractionation, we have identified LexA and two AbrB-like proteins, Sll0359 and Sll0822, which bind to a fragment of the sbtA promoter. Using extracts prepared from LC-grown cells, Sll0822 did not bind to the sbtA promoter despite its presence in the cells, suggesting that it may serve as a repressor of LC-induced genes. This is supported by the fact that sbtA, ndhF3 and cmpA normally expressed only under LC in the wild-type are transcribed under both HC and LC in a Deltasll0822 mutant. When grown under HC this mutant exhibits an elevated apparent photosynthetic affinity to Ci, typically observed in the wild-type only under LC. Clearly, expression of genes essential for Ci uptake was sufficient to raise the apparent photosynthetic affinity for external Ci.

The response of Microcystis aeruginosa strain MGK to a single or two consecutive H2 O2 applications.

Various approaches have been proposed to control/eliminate toxic Microcystis sp. blooms including H2 O2 treatments. Earlier studies showed that pre-exposure of various algae to oxidative stress induced massive cell death when cultures were exposed to an additional H2 O2 treatment. We examined the vulnerability of exponential and stationary-phase Microcystis sp. strain MGK cultures to single and double H2 O2 applications. Stationary cultures show a much higher ability to decompose H2 O2 than younger cultures. Nevertheless, they are more sensitive to an additional H2 O2 dose given 1-6 h after the first one. Transcript analyses following H2 O2 application showed a fast rise in glutathione peroxidase abundance (227-fold within an hour) followed by a steep decline thereafter. Other genes potentially engaged in oxidative stress were far less affected. Metabolic-related genes were downregulated after H2 O2 treatments. Among those examined, the transcript level of prk (encoding phosphoribulose kinase) was the slowest to recover in agreement with the decline in photosynthetic rate revealed by fluorescence measurements. Our findings shed light on the response of Microcystis MGK to oxidative stress suggesting that two consecutive H2 O2 applications of low concentrations are far more effective in controlling Microcystis sp. population than a single dose of a higher concentration.

An AbrB-like protein might be involved in the regulation of cylindrospermopsin production by Aphanizomenon ovalisporum.

Certain filamentous cyanobacteria, including Aphanizomenon ovalisporum, are potentially toxic owing to the formation of the hepatotoxin cylindrospermopsin. We previously identified a gene cluster in A. ovalisporum likely to be involved in cylindrospermopsin biosynthesis, including amidinotransferase (aoaA) and polyketide-synthase (aoaC), transcribed on the reverse strands. Analysis of the genomic region between aoaA and aoaC identified two transcription start points for each of these genes, differentially expressed under nitrogen and light stress conditions. The transcript abundances of these genes and the cylindrospermopsin level were both affected by nitrogen availability and light intensity. Gel shift assays and DNA affinity columns isolated a protein that specifically binds to a 150 bp DNA fragment from the region between aoaA and aoaC, and MS/MS analyses identified similarity to AbrB in other cyanobacteria and in Bacillus sp. Comparison of the native AbrB isolated from A. ovalisporum with that obtained after cloning and overexpression of abrB in Escherichia coli identified specific post-translational modifications in the native cyanobacterial protein. These modifications, which are missing in the protein expressed in E. coli, include N-acetylation and methylation of specific residues. We discuss the possible role of these modifications in the regulation of cylindrospermopsin production in Aphanizomenon.

Enhanced photosynthesis and growth of transgenic plants that express ictB, a gene involved in HCO3- accumulation in cyanobacteria.

Transgenic Arabidopsis thaliana and Nicotiana tabacum plants that express ictB, a gene involved in HCO3- accumulation within the cyanobacterium Synechococcus sp. PCC 7942, exhibited significantly faster photosynthetic rates than the wild-types under limiting but not under saturating CO2 concentrations. Under conditions of low relative humidity, growth of the transgenic A. thaliana plants was considerably faster than the wild-type. This enhancement of growth was not observed under humid conditions. There was no difference in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) detected in the wild-types and their respective transgenic plants. Following activation in vitro, the activities of RubisCO from either low- or high-humidity-grown transgenic plants were similar to those observed in the wild-types. In contrast, the in vivo RubisCO activity, i.e. without prior activation, in plants grown under low humidity was considerably higher in ictB-expressing plants than in their wild-types. The CO2 compensation point in the transgenic plants that express ictB was lower than in the wild-types, suggesting that the concentration of CO2 in close proximity to RubisCO was higher. This may explain the higher activation level of RubisCO and enhanced photosynthetic activities and growth in the transgenic plants. These data indicated a potential use of ictB for the stimulation of crop yield.

Interactions between Scenedesmus and Microcystis may be used to clarify the role of secondary metabolites.

Microcystis sp. are major players in the global intensification of toxic cyanobacterial blooms endangering the water quality of freshwater bodies. A novel green alga identified as Scenedesmus sp., designated strain huji (hereafter S. huji), was isolated from water samples containing toxic Microcystis sp. withdrawn from Lake Kinneret (Sea of Galilee), Israel, suggesting that it produces secondary metabolites that help it withstand the Microcystis toxins. Competition experiments suggested complex interaction between these two organisms and use of spent cell-free media from S. huji caused severe cell lysis in various Microcystis strains. We have isolated active metabolites from the spent S. huji medium. Application of the concentrated allelochemicals interfered with the functionality and perhaps the integrity of the Microcystis cell membrane, as indicated by the rapid effect on the photosynthetic variable fluorescence and leakage of phycobilins and ions. Although some activity was observed towards various bacteria, it did not alter growth of eukaryotic organisms such as the green alga Chlamydomonas reinhardtii.

The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria.

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.