Studies on microcystin and its technology

 

 

We are a group interested in two main goals: factors affecting the expression of genes involved in microcystis synthesis, and role of microcystins in

the cyanobacteria.

 

Toxic cyanobacterial blooms are more and more widespread in surface waters of the earth, and a serious health concern in many areas due to the

production of several toxins, such as high levels of microcystins. Freshwater eutrophyzation has lead to frequent occurrence of blooms. However,

there is widespread debate about the effect of the environment on microcystin production, since the toxicity of blooms can vary between years

under apparently the same environmental conditions.

 

 

Toxic cyanobacterial blooms

 

 

Cyanobacteria produce a broad range of secondary metabolites known as cyanotoxins that have toxic effects on eukaryotes. Among the

cyanotoxins, there is a group of potent hepatoxins called microcystins. Several genera of cyanobacteria, such as Microcystis, Anabaena, Planktothrix,

and Nostoc can produce the cyclic heptapeptide microcystin. Microcystins are potent inhibitors of protein phosphatases 1 and 2A in eukaryotes.

Studies of the regulation and function of cyanotoxins are interconnected and have focused on local ecosystems and the effect of environmental

parameters on toxin production. In nature, cyanobaterial blooms can be unpredictable toxic or non-toxic from one year to the next, and even under

laboratory conditions, results are highly variable. The effects of environmental factors such as light intensity, temperature, nitrogen, phosphorous,

and trace metals on microcystins production have been studied under field and laboratory, and the results are conflicting. There are two main open

questions concerning the microcystins:

1) the regulation of the expression of the genes involved in microcystin synthesis, and the environmental

factors affecting the toxicity of the cyanobacterial populations and

2) the physiological role of microcystis.

 

Structure of microcystin

 

 

FACTORS AFFECTING THE MICROCYSTIN SYNTHESIS

 

  • Results suggest that iron availability is a key factor

 

Although at present the factors unleashing the cyanotoxin synthesis are unknown, iron availavility seems to be a determinant factor in the

expression of the gene cluster involved in the microcystin synthesis (mcy operon). (Martín, et al. Fur from Microcystis aeruginosa binds in

vitro promoter regions of the microcystin biosynthesis gene cluster. Phytochemistry. 67, 876-881. (2006) )

Real time RT-PCR of M. aeruginosa (Sevilla et al., Iron availability affects mcyD expression and microcystin-LR synthesis in Microcystis

aeruginosa PCC7806. Environmental Microbiology, 10, 2476-2483 (2008)), indicated that iron deficiency may induce the expression of mcy

operon.

 

  • Nitrate promote growth, but not microcystin synthesis

 

Our work indicates that M. aeruginosa growth in laboratory conditions in abundance of nitrate in the medium suffer a bloom similar to the

observed in field conditions. But cuantification of microcystin-LR and mcyD expression per cell, indicated that they were not changed during

the bloom. (Sevilla et al., Microcystin-LR synthesis as response to nitrogen: transcriptional analysis of the mcyD gene in Microcystis aeruginosa

PCC7806, Ecotoxicology (2010))

 

  • Light and darkness

 

Microcystin synthesis requires light. In darkness, marked diminution of mcyD expression was detected, similar to the obtained blocking the

photosynthetic electron transfer chain using DCMU.

Also, mcyD expression was induced as consequence of a light shift to high light conditionos.

 

  • Oxidative stress

 

Methyl viologen and hydrogen peroxide decrease the expression of mcyD and microcystin synthesis.

 

 

                 

                                 Liquid culture of Microcystis aeruginosa PCC 7806                         Microcystis aeruginosa PCC 7806 plate culture

 

 

 

ROLE OF MICROCYSTINS IN THE CYANOBATERIA

 

  • Microcystins can bind proteins

In vitro, and probably as consequence of cells disruption, microcystins bind proteins unspecifically. (Vela et al., Exploring the interaction of

microcystin-LR with proteins and DNA. Toxicology in Vitro, 22: 1714-1718 (2008))

 

  • Microcystin-LR can bind some metals

 

EPR experiments indicated that microcystins complex with Cu and Fe 3+

 

 

 

TECHNOLOGY OF MICROCYSTINS

 

In collaboration with the company Zeu-Inmunotec we have developed a simple, rapid in vitro bioassy kit to quantify microcystins. The assay is for the

detection of microcystins and nodularins in water. It is based on the mechanism of action of microcystins on protein phosphatase PP2A. The test

measures the activity of PP2A in water samples potentially contaminated with these toxins. It is therefore an in vitro assay that quantify the toxicity

of all MCs present in the sample and check whether the toxin concentration is over the maximum allowed levels (1 µg/L, WHO 1998).