Gene transfer in bacterial arm races

The following videos are two short documentaries made by students in the MIT Graduate Program in Science Writing. Both films refer to a recent discovery of new antibiotics by scientists at the Massachusetts Institute of Technology and the University of Florida. But, please, don't say: "bah, another antibiotic discovering, so boring". What makes this an interesting story is not the particular antibiotics themselves (we'll see if they ever become useful), but the way they were discovered. Or should we say "created"?

It's been known for some time that the genomes of many microbes contain genes putatively coding for the production of many small molecules. Some of these molecules may have antibiotic, anticancer or other potentially useful activities. By looking at the genomic DNA sequence, scientists can often predict that such a microbe has the potential to produce specific metabolites, belonging to defined structural classes (polyketides, non-ribosomal peptides, glycosides, etc.). However, very often, the predicted metabolites are not detected when the microbe is cultured under standard laboratory conditions. Why is this? The usual explanation is that these molecules are only produced under specific circumstances that the microorganism faces in its natural environment.

With this idea in mind, the above mentioned researchers tried to find antibiotics in the cultures of a bacterium called Rhodococcus fascians (let's call it "Rhodo"). Rhodo belongs to a group of bacteria known as actinomycetes, which are well-known antibiotic producers and whose genomes are rich in information coding for the synthesis of potentially useful metabolites. The scientists cultured the microorganism under a variety of conditions: they tried both standard and unusual ones (starvation, sub-optimal temperatures or culture media, etc.). However, no antibiotic activity was ever detected.

So, they tried to "stress" Rhodo by adding another microbe in the same flasks: the name of the second partner was Streptomyces padanus ("Strepto", for short). Strepto produces a potent antibiotic (actinomycin) that kills bacteria such as Rhodo. So, what was the point? Rhodo died in all flasks, didn't it? Well, not in all of them. In one particular flask (out of hundreds), Rhodo not only survived but actually exterminated Strepto! The "Super-Rhodo" did this by producing a new antibiotic substance, never seen before. Moreover, Super-Rhodo was able to do this thanks to a piece of DNA that Rhodo stole from Strepto!

Both documentaries refer to the same story, but they use remarkably different styles. Please watch both of them, and post a comment if you want to share your thoughts (about them or about the story).

The first video is War in a Petri Dish, by Grace Chua, Allyson Collins, and Lissa Harris:

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The second video is Shot in the Dark, by Andrew Moseman, Rachel VanCott, Megan Rulison, and Ashley Yeager:

Click To Play

There are some scientific aspects that may need some clarification. The initial idea was that Rhodo contained the genetic potential to make antibiotics, and the genes responsible for this were only "switched on" under certain unknown circumstances. This might be correct, but the mentioned results don't clearly validate it. When Rhodo faced a Strepto attack, it simply died. The only survivor (and now a killer itself), Super-Rhodo, had acquired genetic material from its enemy. This extra piece of DNA was essential for production of the new compounds. Although full details have yet to be published, it is not clear if the transferred DNA contained all, or some, of the genes coding for biosynthetic enzymes for antibiotic production. The new antibiotics are not related to actinomycin; however, it is possible that Strepto is able to produce them, in addition to actinomycin (again, under certain unknown circumstances...). Alternatively, the real effect of the extra DNA might be just regulatory, coding for some factor that induced the "switching on" of Rhodo's own genes. We'll wait for the answers to these doubts.

Scholar article:
K. Kurosawa, I. Ghiviriga, T.G. Sambandan, P.A. Lessard, J.E. Barbara, C. Rha, A.J. Sinskey (2008). Rhodostreptomycins, Antibiotics Biosynthesized Following Horizontal Gene Transfer from Streptomyces padanus to Rhodococcus fascians Journal of the American Chemical Society, 130 (4), 1126-1127 DOI: 10.1021/ja077821p

Related link:
Deadly mycelia: predatory streptomycetes. Twisted Bacteria (Jan. 15, 2008).

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An 'open source' approach to drug discovery

Why should we worry about intellectual property protection for infectious diseases and diseases of the poor? Why can't we share our ideas and brains to create an open source platform for drugs for these diseases in the same way that the human genome has been sequenced and the Internet developed?

These are some questions posed by geneticist Samir Brahmachari in an interview published at SciDev.net. It makes an interesting reading.

Samir Brahmachari is the director general of India's Council of Scientific and Industrial Research (CSIR), a network of 38 government laboratories. He is starting an 'open source' approach to drug discovery, starting with tuberculosis (TB). His aim: a system where researchers all over the world work on different areas of drug design and deposit their findings into an open database for others to use and comment on — particularly for infectious diseases that affect the world's poor.

Here I am just gathering a few selected parts from the interview, and adding (see below) a collection of links related to open-source drug discovery.

Excerpts from the interview:

Most public-funded institutions do a lot of biomedical research but the last mile of drug discovery is left to the pharmaceutical industry — which is a 'closed-door' activity.

My idea is that affordable drugs are a right for all, and all drugs can be made available. When it comes to TB or diseases of the poor — where the market incentive is very small — it is not possible to convince the pharmaceutical companies to work on these drugs. Therefore it is the responsibility of public-funded institutions to participate.

Targeted drugs that are market driven — or that rich people can afford — can be made by the [patent-protected] route. But for drugs that are not driven by the market and are needed by the poor, open source is an advantage.

An open source approach runs against the current global emphasis on tightening patents and intellectual property rights. Will it survive opposition from powerful pharmaceutical companies inside and outside India?

On the contrary, you will be surprised to learn that, in the case of TB, many pharmaceutical companies have shown interest in this concept and responded to my initiative. They would like to see a drug breakthrough because of the huge number of patients who need it.

Also, today the private sector is increasingly talking about corporate social responsibility. Private companies are becoming conscious of their social responsibility and many would like to join such initiatives. And there are many private non-profit foundations, such as Bill Gates' Foundation, who support affordable drugs initiatives.

Are you not worried that open source discovery will reduce the incentive for pharmaceutical companies to invest in research, and that public sources will lack the funds to make up the difference?

I am not worried about that. If the private companies do not come forward with research and development in neglected diseases, then it becomes the obligation and responsibility of the public-funded institutes to undertake the research. India is now no longer a poor country and the Indian government can afford to invest money for such research.

Even big pharmaceutical companies, such as AstraZeneca, and leading universities, such as Berkeley, have shown interest in collaboration. Sabeer Bhatia, a founder of Microsoft's Hotmail, has agreed to support us by developing the software.

How applicable is open source to other technological areas?

An important point — somewhat overlooked — is the participation of brilliant minds in the open source model. Where knowledge is free, brilliance flourishes. I believe that, in principle, technology areas such as energy, water and food can also benefit from the open source model. In the case of energy, we may invite solutions to tap solar energy, wind energy, hydropower and other sectors. Similar methods could be adopted in other areas.

When it comes to infectious diseases, compulsory licensing [where pharmaceutical companies must allow their product to be produced cheaply by a country in a medical emergency] should be used on all drug patents so that we can make the drug at low cost and make it affordable for poor people.

The interview is freely available at:
T. V. Padma. Q&A: Advocating open source drugs. SciDev.Net (June 12, 2008).


Links related to open-source drug discovery:

• Open research, Wikipedia.
• Can drug discovery use the open source model? Livemint.com (Sep. 20, 2007).
• Can open-source R&D reinvigorate drug research? Nature Rev. Drug Discovery (2006) 5, 723-729 (freely available at the time of this writing).
• Finding Cures for Tropical Diseases: Is Open Source an Answer? PLoS Med. (2004) 1(3): e56 (open access).
• Open Source Drug Discovery: Finding a Niche (or Maybe Several), SSRN (April 1, 2007).
• Open Source Drug Discovery, X2 (05/02/2008).
• Spicy IP Interview with Dr. Samir K Brahmachari, Spicy IP (March 19, 2008).
• Open Source for Cost Effective Drug Discovery, Spicy IP (Dec. 23, 2007).
• TDI - the Tropical Disease Initiative.
• Open Source Drug Discovery, P2P Foundation.
• Open Source Drug Discovery Foundation.

Image credits: Pill bottle by net_efekt (Christian Guthier). Source: Flickr.

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Merck halts natural products research

Bad news: Merck has decided to close down its natural product research facilities. This means the end of CIBE (Centro de Investigación Básica de España) — or Spanish Center for Biological Research — located in Madrid, Spain. Since its creation in 1954, this center has been dedicated to the discovery of new compounds of therapeutical potential produced by microbes. These efforts led to the development of several useful medicines, including antibiotics (fosfomycin, cefoxitin, thienamycin), cholesterol-lowering drugs (lovastatin), and antifungal agents (caspofungin). In May 2006, Merck researchers hit the news with the discovery of platensimycin, a natural product belonging to a new class of antibiotics.

According to Chemical & Engineering News, the high costs are behind the decision to eliminate natural products research. Merck spokesman Ian R. McConnell explains to C&EN:

"The investment involved in finding these chemicals in the environment is significant. The products that came out of our effort have been significant as well, but that was over a 50-year period"

Sad, but true. Many thousands of natural samples need to be screened in order to detect a bunch of potentially useful compounds, most of which will never become a marketed drug. Turning a promising natural product into a useful medicine takes much effort and time (over 10 years) and, hence, money. So, perhaps it is understandable that most pharmaceutical companies dedicate only a very small fraction of their resources, if any, to natural product drug discovery.

However, even with that little dedication, many medicines in the market have a natural origin, being based in substances originally isolated from plants, microbes, etc. Can we imagine the possible results of investing in natural product research as many resources as those dedicated to chemical synthesis?

Natural compounds often have bizarre, complicated chemical structures and exert their biological effects through unexpected mechanisms. They are the result of an on-going combinatorial chemistry performed by organisms since the origin of life.

Are chemists that good?



Related links

A) About job cuts at Merck:

• Merck cuts sales jobs and halts natural products research. Chemical & Engineering News (May 7, 2008).
• Merck bails on natural products. In the Pipeline (May 8, 2008).
• At Merck, some layoffs are made by PowerPoint. Pharmalot (May 8, 2008).
• Layoffs by PowerPoint? BioJobBlog (May 8, 2008).

B) About CIBE:

• Centro de Investigación Básica de España (CIBE). Merck Sharp & Dohme de España [in Spanish].
• Ten years of CIBE symposia, 1989-1998 (by Sagrario Mochales, CIBE) [pdf]. Int. Microbiol. (1998) 1, 251-254.
• Forty years of screening programmes for antibiotics (by S. Mochales). Microbiología (1994) 10, 331-342.
• Declaraciones de Fernando Peláez, director del CIBE, durante la presentación del BIO-Europe Spring (Marzo 2008) [video, in Spanish].
• Este pueblo puede salvar a España del colesterol (incluye una entrevista a Sagrario Mochales, "la científica olvidada"). ElMundo.es (16 Dic. 2007) [in Spanish].
• El tesoro de la selva tropical llega a Madrid. ElPaís.com (14 Jun. 1995) [in Spanish].

C) About natural products in drug discovery:

• Natural products to drugs: natural product-derived compounds in clinical trials. Nat. Prod. Rep. (2008) 25, 475-516.
• Natural products: chemical instruments to apprehend biological symphony. Org. Biomol. Chem. (2008) 6, 424-432.
• Natural products as sources of new drugs over the last 25 years. J. Nat. Prod. (2007) 70, 461-477.
• New aspects of natural products in drug discovery. Trends Microbiol. (2007) 15, 279-289.
• The value of natural products to future pharmaceutical discovery. Nat. Prod. Rep. (2007) 24, 1225-1244.
• Natural product drug discovery: the times have never been better. Chem. Biol. (2007) 14, 1098-1104.
• The historical delivery of antibiotics from microbial natural products--can history repeat? (By Fernando Peláez, CIBE). Biochem. Pharmacol. (2006) 71, 981-990.
• Drug discovery from natural sources. AAPS Journal (2006) 8, E239-E253.
• Antibacterial natural products in medicinal chemistry--exodus or revival? Angew. Chem. Int. Ed. Engl. (2006) 45, 5072-5129.
• The renaissance of natural products as drug candidates. Science (2005) 310, 451-453.
• The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. (2005) 4, 206-220.
• Rediscovering natural products. Chemical & Engineering News (Oct. 13, 2003).

Image credits:
Digital Atlas of Actinomycetes. Copyright: Society for Actinomycetes Japan. Contributor: S. Mochales. This strain produces β-lactam antibiotics, thienamycins. It has the color of cattleya orchids.

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Women scientists, sixty years ago

New York City, 1949. During the last three years, Elizabeth Hazen had been isolating hundreds of microbes from dirt samples taken at different locations. Many microbiologists at the time were following a path open by Alexander Fleming, Selman Waksman and others, who discovered that some soil microbes produced certain substances—antibiotics—with powerful activities against bacteria. However, rather than looking for a new agent against prokaryotic microbes, Elizabeth searched for a medicine to fight fungal infections. For this purpose, she grew the soil microbes and tested the cultures against disease-causing fungi (Cryptococcus neoformans, Candida albicans [see image]). Whenever a culture showed an interesting activity, she put it in a glass Mason jar and mailed it to Albany, 250-km away. Here, Rachel Brown—a chemist—used the culture for purification and characterization of the active compound. Then, Rachel mailed the fruit of her efforts back to New York, where the microbiologist tested the sample again for fungicidal potency. Through this collaboration, the two scientists isolated several antifungal compounds that, unfortunately, were too toxic when tested in laboratory animals.

But, finally, Elizabeth and Rachel found a useful fungicidal agent with a lower toxicity. It was produced by a soil bacterium isolated from a sample that Elizabeth had collected, while on holiday, in Warranton, Virginia. She had taken a bit of soil at the edge of a cow pasture, near a dairy barn, at the farm of a certain Walter B. Nourse. Because the microbe appeared to be a new species of streptomycetes, it received the name Streptomyces noursei, in honor of Mr. Nourse. The fungicidal agent was initially named fungicidin, but it was soon renamed nystatin, as both Elizabeth and Rachel worked for the New York State Department of Health (although in different locations). Since then, nystatin has been widely used to treat candidiasis and other fungal infections.


Related links:

• Elizabeht Hazen, seeker. Arts of Innovation.
• Elizabeth Hazen and Rachel Fuller Brown, Pharmaceutical Achievers. Antibiotics in Action.
• News of Science: Scientists in the News (on E. Hazen). Science (1955) 122, 914.
• Women in Microbiology, Microbes and Society: A Closer Look.
• Elizabeth Hazen Biography. World of Microbiology and Immunology.
• Elizabeth Lee Hazen and Rachel Fuller Brown. Inventor of the Week, Lemelson-MIT Program.

This post modestly celebrates March 8th, International Women's Day. The discovery of nystatin seems a good example of an important contribution of women scientists to microbiology, natural product chemistry, and medicine. A related story is that of Alma Whiffen, who discovered cycloheximide—also known as actidione—around the same time (1947). She isolated the compound from cultures of a soil microbe, Streptomyces griseus. Cycloheximide has antifungal activity, and was employed to treat fungal infections in plants; however, it is not useful for human treatment. The compound is better known as a general inhibitor of protein synthesis in eukaryotes, and it is widely used for research purposes. Read more here:

• Antibiotic for Plants. Time (Nov. 22, 1948).
• Alma Whiffen Barksdale Records. The New York Botanical Garden.

More related links:

• Microbiology pioneer dies (Esther Lederberg, discoverer of the lambda phage). Aetiology (Nov. 30, 2006).
• Committee on the Status of Women in Microbiology, American Society for Microbiology.
  
• Women in Science, the blog.
• Iraq's women scientists. BBC News, Middle East (Sept. 22, 2004). The dark side of microbiology...

Image credits: Wikipedia.

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Finding a needle in the ocean

The deep ocean may be similar to a rainforest in terms of the range of existing microbes and their genetic diversity. The resulting biochemical diversity might provide us with novel natural drugs and enzymes for cleaner industrial processes. The following clips are available for download from Out of the Blue, a DVD on marine microbes produced by Panache Productions with support from the NERC BlueMicrobe knowledge transfer network. The interviewed researchers are Alan Bull and Jem Stach, from the Universities of Kent and Newcastle (UK), respectively. For bioprospecting, the researchers use a combination of molecular techniques, bioinformatics, novel culturing strategies and screening approaches. In addition to potential pharmaceutical and biotechnological applications, these efforts will broaden our knowledge of microbial ecology and evolution (scarce knowledge, by the way).



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This post is a contribution to Microbial Week, a collection of posts highlighting the many roles of microbes in deep-sea or marine environments. The event is organized by Christina Kellogg and the guys at Deep-Sea News.
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From Aladdin’s Cave to Treasure Island

For a long time, it was thought that the land of actinomycetes was... well, land. I mean, they were supposed to be terrestrial creatures, even although some of them were isolated from samples taken in sea habitats (for instance, read this article from 1958). But these "marine" bacteria, generally found in shallow waters, were quite similar to their counterparts from land. For this reason, it was assumed that any actinomycetes obtained from the sea were just wash-offs from the shore.

Now this view is changing. But how can we say if a microbe isolated from a particular sea location is a true neighbor on the block (as opposed to be just derived from a passing-by or dormant spore, coming from land)? Ideally, it should be recognized by the following criteria: its ability to grow optimally at native conditions (salinity, pressure, temperature, nutrients); demonstration that the organism is really active on location; and the recognition of particular metabolic profiles, not found in terrestrial relatives. Nowadays, the existence of truly marine actinomycetes seems to be supported by solid data.

Similarly to their terrestrial relatives, marine microbes are a rich source of bioactive metabolites (antibiotics, antitumor drugs) and enzymes with different applications. For instance, cultivation of a marine actinomycete known as Salinispora tropica yielded a number of novel metabolites, not found before. One of these compounds, salinosporamide A, has antitumor properties and is currently being tested in humans for the treatment of cancer. Sequencing the genome of Salinispora tropica unveiled a number of genes coding for the synthesis of 17 potential metabolites; most of these compounds had not been detected in previous culturing of the microbe. Then, the researchers used the genetic information to guide a new chemical analysis of Salinispora cultures. The analysis uncovered an additional, novel compound (salinilactam), which had a structure corresponding to that deduced from the DNA sequence.

Let me finish with David Hopwood's words from Therapeutic treasures from the deep:

"In a recent book I likened the plethora of previously unknown genes in a newly sequenced Streptomyces genome to an Aladdin's Cave. Perhaps Treasure Island would be a more apt metaphor in this case [Salinispora]"

Neat.


List of links:
- Article from 1958: Grein A, Meyers SP, Growth characteristics and antibiotic production of actinomycetes isolated from littoral sediments and materials suspended in sea water. J. Bacteriol. 1958, 76, 457-463.
- Marine actinomycetes: Bull AT, Stach JE, Ward AC, Goodfellow M, Marine actinobacteria: perspectives, challenges, future directions. Antonie Van Leeuwenhoek, 2005, 87, 65-79.
- Salinosporamide A: Wikipedia.
- Sequencing the genome of Salinispora tropica: Udwary DW, Zeigler L, Asolkar RN, Singan V, Lapidus A, Fenical W, Jensen PR, Moore BS, Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica. Proc. Natl. Acad. Sci. USA, 2007 , 104, 10376-10381.
- Therapeutic treasures from the deep: Hopwood DA, Nat. Chem. Biol. 2007, 3, 457-458.
- Aladdin's Cave: Aladdin - Wikipedia.
- Treasure Island: Wikipedia.


Images:
Left, Aladdin in the Magic Garden, an illustration by Max Liebert for Ludwig Fulda's Aladin und die Wunderlampe. Source: Wikipedia.
Right, Jim Hawkins and the treasure of Treasure Island, an illustration by Georges Roux for the 1885 edition of Treasure Island by Robert Louis Stevenson. Source: Wikipedia.

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This post is my contribution to Microbial Week, a collection of posts highlighting the many roles of microbes in deep-sea or marine environments. The event is organized by Christina Kellogg and the guys at Deep-Sea News.
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Yondelis: from the seas to the pharmacy

Yondelis (trabectedin, ecteinascidin-743, ET-743) is an anticancer natural product isolated from a marine organism, the tunicate Ecteinascidia turbinata. The compound was initially extracted from the naturally growing and farmed tunicate, but now it's manufactured by semi-synthesis from a structurally-related metabolite produced by the bacterium Pseudomonas fluorescens. It is likely that the actual producer of Yondelis is not the tunicate itself but some unknown, symbiotic bacteria living in close relationship to the tunicate. The drug is being developed by PharmaMar, a Spanish biopharmaceutical company subsidiary of the Zeltia Group. For several years, Yondelis has being studied in clinical trials for treatment of several cancer types.

This morning, finally, good news: the European Medicines Agency has recommended Yondelis be approved by the European Comission, which means that the drug should be available to treat soft tissue sarcomas by the end of the year. I want to cite the words of José María Fernández Sousa (president of Zeltia), taken from the PharmaMar press release:

“This is excellent news. Firstly for the patients and their families, since a new therapeutic option is now available. Also for the PharmaMar and Zeltia employees who have devoted long years of effort and dedication in pursuit of this challenge. It is also excellent news for Spanish science as well as for the investigators from all over the world who have participated in the clinical trials and believed in the therapeutic potential of compounds of marine origin.”

I completely agree with him. Yondelis is the first anticancer drug developed and produced by a Spanish biopharmaceutical company. Hope more are coming.

(Image: left, a cluster of Ecteinascidia turbinata, photo courtesy of PharmaMar; right, structure of Yondelis)

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Combinatorial biosynthesis, but not as we know it

Combinatorial biosynthesis can be understood as a special case of metabolic engineering, where genes responsible for individual metabolic reactions from different organisms are combined to generate hybrid metabolic pathways. An ideal result consists of a genetically-modified organism (or a collection of such microorganisms) that is useful for in vivo production of novel compounds. Nevertheless, there are examples of in vitro combinatorial biosynthesis (*), such as glycorandomization (a biocatalytic technique that uses purified enzymes to activate and attach sugars to natural products).

One step beyond, a recent report in ACS Chemical Biology describes a new in vitro approach for the generation of combinatorial libraries of compounds derived from natural products. As a proof of concept, the authors combined three type-III polyketide synthases (PKSs), 16 different precursors (acyl-CoA esters) and three post-PKS tailoring enzymes. Remarkably, this strategy was adapted to a convenient microarray format (30-nanoliter reactions), to enable high-throughput synthesis. Even better, the same microarray slide was used to screen for bioactivity of the synthesized products, through an assay for inhibition of human protein kinase FynT. This approach is, therefore, potentially useful for the identification of new non-natural compounds displaying biological activities.

Reference: Kwon SJ, Lee My, Ku B, Sherman DH, Dordick JS.
High-throughput, microarray-based synthesis of natural product analogues via in vitro metabolic pathway construction.
ACS Chem. Biol. 2007 May 25 (ASAP Article). PubMed link.

[(*) I understand that some people may prefer to keep the term "combinatorial biosynthesis" only for in vivo approaches. Perhaps they're right, but I view biocatalysis (in vitro utilization of purified enzymes for chemical transformations) as a special type of biosynthesis.]

[Another note: I'm not a Star-Trek fan, but I think that the line "It's life, Jim, but not as we know it" was told by the Doc to the captain on seeing new life on a strange planet. However, I didn't learn the line directly from Star Trek, but from an article titled "Life, Jim, but not as we know it"? Transmissible dementias and the prion protein, Br. J. Psychiatry (1991) 158: 457-4710, authored by PJ Harrison & GW Roberts. I got to know this article while learning about neurodegenerative diseases during my M.S. studies (uf, feels like late Pleistocene). It was such a great title for a story on prions, it just stuck in my mind.]

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