Analysis of changes over time in microcystin content of nitrogen-limited Microcystis aeruginosa (Kützing) Lemmermann batch cultures (strain MASHOl-A19) showed that net microcystin production was limited to the phase of growth when cell concentration was increasing. The net microcystin production rate decreased as the specific cell division rate (µc) decreased, but, more importantly, the specific toxin production rate (µMCYST) decreased at an identical rate to that of µc when the culture became nitrate-limited. The actual size of the microcystin pool (total culture microcystin concentration) increased while cells were dividing, then remained constant or decreased only slightly during the stationary and death phases, even when the cultures were severely nitrate-starved. These findings demonstrate conclusively that the processes of cell division and microcystin production are tightly coupled under nitrogen-limited cell division. Our findings suggest that microcystin production is controlled by environmental effects on the rate of cell division, not through any direct effect on the metabolic pathways of toxin production. Reevaluation of data presented by others shows this to be the case for two other cyanobacterial species producing nine different microcystins over a wide range of environmental variables. We believe these relationships now provide a unifying view of environmental control of microcystin production in hepatotoxic cyanobacteria. We conclude that there is a direct linear correlation between cell division and microcystin production rates in all microcystin-producing cyanobacteria regardless of the environmental factor that is limiting cell division. We also conclude that microcystin is not a secondary metabolite, as is currently thought, but that it displays many of the attributes of essential intracellular nitrogenous compounds in toxigenic cyanobacteria.Microcystis aeruginosa (Kützing) Lemmermann is one of a number of species of cyanobacteria that may produce a suite of cyclic peptide hepatotoxins known as microcystins (Botes et al. 1984; Carmichael 1994). Because of growing public health concerns, there has been intense interest by scientists and water managers in elucidating the factors controlling the toxicity of blooms of M. aeruginosa and other cyanobacterial species (Carmichael 1992a(Carmichael , 1994.The gravimetric microcystin (MCYST) concentration of dried M. aeruginosa may vary by more than three orders of magnitude between individual bloom and culture isolates (Belch et al. 1997). Indeed, many isolates and blooms are reported to be nontoxic, reputedly containing no microcystin. The problem facing researchers is to ascertain whether the observed variation in microcystin content of field samples results from changes in the dominance of strains of varying microcystin content or from the influence of changing environmental variables on microcystin synthesis and metabolism. The literature provides evidence to support both mechanisms in culture and natural waters. Genetic variation is mos...
Cell quotas of microcystin (Q MCYST ; femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q MCYST and specific growth rate (), from which we propose a generalized model that enables Q MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q MCYST from , max (maximum specific growth rate), Q MCYSTmax (maximum cell quota), and Q MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q MCYSTmax of 0.129 fmol cell ؊1 at max and a Q MCYSTmin of 0.050 fmol cell ؊1 at ؍ 0. Net MCYST production rate (R MCYST ) asymptotes to zero at ؍ 0 and reaches a maximum of 0.155 fmol cell The microcystins (MCYSTs) are a group of cyclic heptapeptide toxins produced by several cyanobacterial species. Of the more than 60 MCYSTs characterized to date (19,27,29), most are potent inhibitors of protein phosphatases 1 and 2A from both plants and animals (17). One of the most common MCYST-producing cyanobacteria is the bloom-forming Microcystis aeruginosa (Kützing) Lemmermann. Due to the widespread distribution and potential toxicity of this species (toxic strains have been found worldwide), M. aeruginosa has been implicated in a number of animal-poisoning incidents (e.g., reference 7) and more recently in human fatalities (11,23).M. aeruginosa is a unicellular, colonial freshwater cyanobacterium which often forms blooms during warmer months in eutrophic lakes and reservoirs (37). For this reason, much research has been concerned with the environmental factors which lead to bloom formation and toxin production in this species. A wide range of batch culture studies have shown that the variables influencing MCYST content include trace metal supply (15), nitrogen (N) and phosphorus (P) (31), light and temperature (38), and pH (34). Comparative studies on MCYST production by M. aeruginosa in continuous culture, however, have been limited to examination of the effects of photon irradiance (35), N, P, and Fe 3ϩ limitation (16, 36), and more recently P limitation (20). Despite this considerable pool of data concerning MCYST production, few studies (with the exception of the work carried out by Rapala and coworkers [25,26]) have been able to quantitatively relate MCYST content to any growth determinant.In a previous batch culture study, we presented data on the effect of N supply on the cellular production of MCYSTs (21). This work showed that the net specific rate of MCYST production was equal to the cell specific growth rate. The application of these findings to previously published batch culture studies suggested that the relationship held under a variety of culture conditions and that MCYST production was indirectly affected by environmental factors through their effects on cell division. A consequence of this l...
An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH 2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu--methylaspartate-Arg-OH, and the other is the tetrapeptide NH 2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 g/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.
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