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An ingenious trick of nature: bacterial toxin-antitoxin systems

Cyanobacteria, also known as blue-green algae, are the oldest known form of life and have been around for 3 billion years. It stands to reason therefore that they should be relatively simple and primitive organisms. But this is not quite the case: two scientists from the Institute of Biology III at Freiburg University, Stefan Kopfmann and Prof. Dr. Wolfgang Hess, have discovered that cyanobacteria have developed a clever natural selection mechanism. Amongst other things, this mechanism ensures the survival of the cyanobacterial colony, even if this means that some individuals have to ‘commit suicide’.

There are around 2000 different types of cyanobacteria, all of which use the energy of sunlight to drive photosynthesis. Cyanobacteria are the major producers of oxygen in the atmosphere, which is why they were previously regarded as algae. “We are extremely interested in Synechocystis, especially as these cyanobacteria can live in both freshwater and salt water and have also been found in deserts,” says Stefan Kopfmann, doctoral student in the Hess laboratory. “Moreover, some of the strains are really quite extraordinary and can even fix nitrogen,” says Kopfmann referring to one of Synechocystis’ unusual abilities. Synechocystis cells also occasionally commit suicide. This alone sounds like a great plot for a Hollywood film, but add to that the fact that the deaths are the result of a toxin produced by the victims themselves, and you have even more drama.

Is the bacteria’s suicide of any use?

The bacterial strain Synechocystis PCC6803; left: microscope image; right: autofluorescence. © Stefan Kopfmann, University of Freiburg

Like other bacteria, cyanobacteria have no nucleus. Instead, they contain a central nucleoid that has many fine strands of DNA. Moreover, cyanobacteria have extrachromosomal elements, i.e. plasmids which also carry other genetic information. However, this information is not essential for the bacteria’s primary functions, i.e. breathing, carrying out photosynthesis and reproduction. So the question arises as to why cyanobacteria have plasmids at all. 

Like other living organisms, bacteria exchange information with one another. They do so in the form of genetic information that is encoded on plasmids. The bacterial exchange of genetic information is known as horizontal gene transfer, a process that is common among bacteria, even amongst very distantly related ones, and happens relatively rapidly. The cyanobacterial plasmid also harbours genes that encode a rather sophisticated poisoning system. These genes are activated under certain circumstances and result in the death of the affected bacterial cell. 

For multicellular organisms, the process of programmed cell death (apoptosis) has a protective function and is therefore a logical thing to do. So the question arises as to why single-celled organisms commit suicide and die. “This does not seem to make sense, at least not at first sight,” said Wolfgang Hess. “Why would a cell produce a toxin as well as an antitoxin to neutralise the poisonous effect of the first one?” 

Toxin and antitoxin as safety system

Toxin-antitoxin systems that are contained on plasmids ensure that daughter cells that inherit the plasmid survive after cell division. If the plasmid is absent in a daughter cell, the stable toxin kills the new cell. © Stefan Kopfmann, University of Freiburg
The toxin-antitoxin system found in Synechocystis is quite a common phenomenon in bacteria; it is often crucial for the survival of the entire bacterial population. Back in the 1980s, researchers discovered that the genes for toxins and antitoxins are located together on a plasmid. The cells therefore produce them together, resulting in the fact that the antitoxin neutralises the toxin. They also found that the toxin was more stable than the antitoxin and therefore effective for a longer period of time. The cell needs to constantly replenish the antitoxin in order to survive. Bacterial cells possess numerous plasmids, which are distributed randomly to the daughter cells. In consequence, when a cell loses the plasmid during cell division, both genes are lost. “Cells without these particular plasmids are unable to produce toxins and antitoxins. Since the toxin is more stable than the antitoxin and is thus effective for a longer period of time, these cells eventually die off,” said Hess explaining the sophisticated toxin-antitoxin system. Toxin-antitoxin systems on plasmids can fulfil several functions. However, as mentioned above, organisms in which the plasmid is absent die. It appears that the plasmid ‘knows’ how to protect itself. This all seems rather plausible as the plasmid also contains genetic information for other functions. “The plasmid has a very special extra function and the bacteria are careful to retain this function,” said Hess referring to the fact that the Synechocystis plasmid also possesses the genetic information for a prokaryotic immune system.

Cyanobacteria have an immune system

This bacterial immune system discovered in 2008 works of course very differently from the mammalian one although it is just as effective. Bacteria need to protect themselves against bacteriophages, viruses that infect and replicate in bacteria. Without its bacterial immune system, the phage-infected cell would die slowly but surely, during which time the phage would continue to replicate and infect numerous new cells. 

Bacteria possess so-called CRISPRs (clustered regularly interspaced short palindromic repeats), genetic information rather than antibodies. CRISPRs function as a prokaryotic immune system. Upon primary infection with a virus, short segments of DNA are incorporated into the bacterial genome between CRISPR repeats and serve as a ‘memory’ of past exposures. The CRISPR arrays are transcribed and processed into shorter RNA molecules. Upon secondary exposure to a virus, the RNA molecules within a ribonucleoprotein complex serve as guides to target invasive viral molecules in a sequence-dependent manner. Specific enzymes then cleave the virulent information, resulting in its degradation before it can cause damage. Hundreds of such phage segments are found in every CRISPR system and are passed on to daughter cells over thousands of years by way of cell division. 

Bacteria are under enormous selection pressure due to the estimated number of over a sextillion phages. “Synechocystis has been cultivated in the laboratory since 1969. Although these cell cultures have never been exposed to phages, the bacterial immune system has remained unchanged ever since,” Hess says, going on to add, “and this can now be explained with the bacterial toxin-antitoxin system.”

Cyanobacteria research is booming

Kopfmann discovered that the Synechocystis plasmid has seven systems of this kind and is thus well protected. “This shows how important the extrachromosomal plasmid is,” the scientist concludes. One of these is toxin-antitoxin pairs is VAP-B/C (VAP: virulence associated protein), wherein C encodes a toxin (an RNA interferase) and B a matching antitoxin that targets C, which – if it is not inhibited – degrades the RNA and the bacterium dies.

These findings therefore provide interesting insights into potential ways of combating bacteria. “A drug that targets the bacterial antitoxin would be an effective way of killing bacterial cells; this would be a rather elegant way to drive the bacteria to suicide,” said Hess.

The commercial interest in cyanobacteria has also increased rapidly over the past years. As was recently announced, the small blue-green organisms possess alcohol dehydrogenases, enzymes that fulfil a variety of functions and are also key enzymes in ethanol generation by bacteria. This is a new form of biotechnology and moreover, one of huge ecological relevance. “Cyanobacteria can be harnessed to convert solar energy directly into biofuels. The use of cyanobacteria for generating biofuels is cheaper than existing methods, requires much less energy and is not in competition with food production,” Hess concludes.

Further information:

Prof. Dr. Wolfgang Hess
Stefan Kopfmann
Genetics and Experimental Bioinformatics
Institute of Biology III III
University of Freiburg
Schänzlestr. 1
79104 Freiburg
Tel.: +49 (0)761/203-2796
Fax: +49 (0)761/203-2601
E-mail: Wolfgang.Hess(at)biologie.uni-freiburg.de

Website address: https://www.biooekonomie-bw.de/en/articles/news/an-ingenious-trick-of-nature-bacterial-toxin-antitoxin-systems