Thaxtomin, a next-generation weed killer ?

It is called “Streptomyces scabies”. This bacterium is feared by farmers because it causes common potato scab, a disease which despite being harmless causes distasteful lesions to appear on tuber crops. And yet this bacterium could become a micro-scale helper for these same farmers because the toxin it produces, thaxtomin, is a natural and biodegradable herbicide which is to date unfortunately too expensive to produce on a grand scale. Two researchers at the Center of Protein Engineering at the University of Liege, Sébastien Rigali and Samuel Jourdan, in collaboration with Dr. Isolde Francis and Professor Rosemary Loria of the University of Florida, have just discovered the molecular mechanism responsible for inducing the synthesis of this toxin. Their discovery forms the basis of a patent proposal whose objective is to make thaxtomin as a commercially viable natural herbicide.

The appearance of these bacteria is not their strong point. Causing brownish scab lesions on all or some of the skin of tubers they do not look very appetizing. Even the name of the disease is enough to cause disgust. After all, who would want to eat potatoes affected by common scab?

Yet despite the unsavory appearance and name of the disease affecting these potatoes, they are not unsuitable for consumption. However, in our societies where appearance counts, we rarely find tubers affected by the disease on sale in supermarkets. This is a great pity considering the staggering losses that the removal of these tubers from supermarket shelves can cause. In Canada, in 2007, researchers estimated annual losses related to the disease at between 15 and 18 million dollars. No data is available for Belgium but common scab has no borders...

The main causative agent of this disease is a bacterium called Streptomyces scabies. It is the black sheep of its family in some respects: among the hundreds of different species of Streptomyces, the latter is one of the rare pathogenic members, that is to say, capable of causing disease in the plant concerned. The potato is not its only victim; it also affects carrots, beetroot, turnips and radishes... It is somewhat out of tune with its many “cousins” which by way of contrast are reputed for their beneficial effects. They are, in fact, the source for more than half of the natural antibiotics, anticancer, antibacterial and antifungal products used in human medicine or in the agriculture industry.  

The tip of the iceberg

It is no surprise that these Streptomyces should be in the sights of Sébastien Rigali and Samuel Jourdan, both of whom are researchers at the Center of Protein Engineering (C.I.P.) at ULg. “Our focus in the laboratory is to understand when, how and why these bacteria produce a molecule that has an antibiotic activity, the meaning of antibiotic being ‘against life’: against viruses, bacteria, plants, fungi, etc. The purpose of this is to find new antibiotics. We now know that the 10,000 molecules of Streptomyces that have been isolated to date are merely the tip of the iceberg! Genome mining has revealed the existence of numerous ‘cryptic’ antibiotics, that is to say, they are unknown because they are not produced under laboratory culture conditions. The genes coding for the proteins that synthesize these cryptic molecules are often ‘silent’, that is to say barely expressed or not at all. The key to unlocking the system must be found. These keys are the transcriptional regulators, proteins that are capable of repressing or activating the expression of genes”.

Scientists have been tirelessly attempting to identify the keys and locks involved in the expression of the genes involved in the production of antibiotics. Once the lock-and-key systems have been decoded, it becomes possible to identify the elicitors of the system, that is to say the triggering elements which, in their natural environment, cause the bacteria to produce these cryptic antibiotics. You might as well be looking for a needle in a haystack given the infinite number of lock-and-key combinations and elicitors that exist!  

At ULg, in direct collaboration with the Universities of  Leiden (NL) and Erlangen (DE), Sébastien Rigali  was the first, in 2008, to identify a complete signaling cascade (keys, locks, and elicitor) in Streptomyces coelicolor, from the extracellular perception of a signal to its transformation into a decision-making role: the production of an antibiotic (1). Since then, the researchers in the ‘Streptomyces Genetics and Development’ group of C.I.P. have been trying to repeat this for other Streptomyces. To succeed, they plan to adopt a bioinformatics approach by using the PREDetector program (2) which makes it possible to detect the ‘locks’ involved in gene expression. Once these locks have been detected, it will be easier to find the key and then identify the environmental elicitor for the system.

A Belgian tale

It was Christmas Eve 2012, when suddenly, bingo! Just as preparations were being made for the annual festivities, PREDetector detected DNA motifs (locks) recognized by the transcriptional repressor (the key) associated with the thaxtomin-synthesizing genes in Streptomyces scabies. It is this molecule that allows S. scabies to attack the roots and tubers of plants in order to extract their nutritional reserves and which is behind these horrible brown-tinted craters on the potatoes.

On returning to the laboratory, Samuel Jourdan was informed about the PREDetector predictions and few weeks later, the latter confirmed that the key which fits the lock is indeed CebR, the transcription repressor for the use of cellulose. The researchers from Liege decided to share their discovery with Prof. Rosemary Loria of the University of Florida in Gainesville (USA), a pioneer in this area of research for about 20 years. In Gainesville, another Belgian, Dr. Isolde Francis, was awarded a post-doctoral grant for the specific study of the induction mechanisms involved in the production of thaxtomin in S. scabies.

“Revealing results that are unpublished is an unusual practice in the realm of science. In general, individuals prefer to keep their discoveries to themselves, partly out of fear of being cheated but also because any public disclosure eliminates the possibility of filing a patent.  Nevertheless, we have always contacted the main interested parties in order to comment upon and share our work and this applies to all our research subjects. Not to contact Rosemary Loria would have been a professional mistake! She is the most important specialist in this subject; we were delighted to work with her”.
 
There is therefore a perfect understanding between the Belgian researchers in Liege and those in Gainesville, and the work is equitably shared: the in vitro experiments are conducted in Liege and the in vivo approaches in Florida. They quickly discovered the way in which S. scabies triggers its pathogenic behavior and generates a series of mutants that produce high quantities of thaxtomin, or that, conversely, do not produce thaxtomin at all.

A double locking system

How does it work? “S. scabies has to detect the presence of its host in the environment. Without perception, there is no production of thaxtomin and therefore no attack on the plants”, observes Samuel Jourdan. “With our colleagues from the University of Florida we discovered how this signal is perceived by Streptomyces scabies and how this triggers the production of the phytotoxin”.

But what is this signal? Rosemary Loria and her colleagues had already found the environmental elicitors for thaxtomin in 2007, those which act as the ‘starting pistol’ for the production of the toxin (3). The molecules which give the ‘green light’, are cellobiose and cellotriose (dimers and trimers of glucose respectively), that is to say, disaccharide and trisaccharide components of cellulose that are found in the walls of plant cells (4).

“In response to cellobiose or cellotriose emanating from the walls of the plant cells, CebR no longer represses the expression of the genes that code not only for the production of thaxtomin, but also for a gene called txtR, which codes for an activator for the expression of these same synthesis genes. We are therefore in the presence of a locking system with two keys: one key which closes called CebR, which controls a key that opens called TxtR. When the cellobiose or cellotriose fixes on CebR, the latter can no longer prevent the expression of the key that opens, TxtR. The inactivation of the cebR gene results therefore in a constant expression of TxtR, and S. scabies presents a hypervirulent behavior because it excessively produces phytotoxin”, explains Sébastien Rigali (opposite).

In other words, once CebR is neutralized, once it has been withdrawn from the chromosome, Streptomyces scabies starts to produce thaxtomin constitutively, that is to say, without needing to perceive the external triggering signal any longer (cellobiose or cellotriose). The result is visible to the naked eye. The cultures of the wild-type strain of S. scabies do not produce thaxtomin (yellow-pigmented), while the culture prepared with the strain of S. scabies where the cebR gene has been inactivated is completely yellow!

A new herbicide onto the market?

Thaxtomin has the particularity of attacking the enzyme which synthetizes cellulose in plants and, consequently, prevents them from growing. In other words, it is a powerful weed-killer. It has the added advantage of being biodegradable: soils already contain microorganisms that are capable of using them as a source of nutrition (5). Thaxtomin, as a biopesticide, offers an interesting alternative to chemically synthesized pesticides. Several companies hold patents for the exploitation of this molecule but marketing thaxtomin is not very profitable because the cost of producing it is very high. A detailed consultation with the suppliers of this molecule revealed that one thousandth of one gramme currently costs between 260 and 590 Euros.

The discovery made by Sébastien Rigali, Samuel Jourdan and their colleagues at the University of Florida is a game-changer because, thanks to this genetic manipulation involving removal of the famous closing key, the mutant bacteria massively produce thaxtomin, or ‘constitutively’, as it is described in the jargon. They are now able to produce grams of thaxtomin at cost-effective industrial scale.

Streptomyces scabies is therefore nothing like a black sheep…after one genetic modification! Their findings will do nothing to prevent the appearance of common scab: potatoes will remain covered with these scab-like lesions.  “Potatoes covered with scab lesions are eatable. It is up to people to change their consumption habits”!  It would undoubtedly be an easier task to stop buying with one’s eyes. Their research has just been published (6) in mBio, an online journal which is a reference in microbiology matters. Furthermore, their work has just been recommended in F1000Prime as being of special significance in the field of pathogenic microbiology. Although the two researchers from Liege have no intention of transforming themselves into thaxtomin dealers, a patent has been filed in collaboration with the University of Florida.

“That we made thaxtomin exploitation possible at an industrial scale is a great step forward for the herbicide to become commercially viable. Our findings come right on time as glyphosate, the most used chemical herbicide notably under the trade name Roundup, has recently been predicted to cause cancer. Investigations on the toxicity of thaxtomin still have to be performed. However, because thaxtomin is naturally produced in the soil, plants and animal species have learned to live with this biomolecule and, importantly, the microorganisms neighboring S. scabies had hundreds of millions of years to exactly know how to degrade this compound. We therefore have many reasons to believe that, when the use of an herbicidal agent will be necessary, this next-generation natural phytotoxin should alter the environment as little as possible”. 

(1) Rigali S, Titgemeyer F, Barends S, Mulder S, Thomae AW, Hopwood DA, van Wezel GP. 2008. Feast or famine: the global regulator DasR links nutrient stress to antibiotic production by Streptomyces. EMBO reports 9:670-675.
(2) Hiard S, Maree R, Colson S, Hoskisson PA, Titgemeyer F, van Wezel GP, Joris B, Wehenkel L, Rigali S. 2007. PREDetector: a new tool to identify regulatory elements in bacterial genomes. Biochemical and biophysical research communications 357:861-864

(3) Wach MJ, Krasnoff SB, Loria R, Gibson DM. 2007. Effect of carbohydrates on the production of thaxtomin A by Streptomyces acidiscabies. Archives of microbiology 188:81-88.
(4) Johnson EG, Joshi MV, Gibson DM, Loria R. 2007. Cello-oligosaccharides released from host plants induce pathogenicity in scab-causing Streptomyces species. Physiological and Molecular Plant Pathology 71:18-25.

(5) Doumbou CL, Akimov VV, Beaulieu C. 1998. Selection and characterization of microorganisms utilizing thaxtomin A, a phytotoxin produced by streptomyces scabies. Applied and environmental microbiology 64:4313-4316.
(6) The Cellobiose Sensor CebR is the Gatekeeper of Streptomyces scabies Pathogenicity, Francis and Jourdan et al. mBio, 2015.