Pioneering research on Bacillus subtilis metabolism reveals bacterium's secrets

Though it lives naturally in the soil, the bacterium Bacillus subtilis is widely used as a model laboratory organism. It is also used as a 'cell factory' to produce vitamins for the food industry and, in biotechnology, to produce enzymes such as those used in washing powders.

The BaSysBio research project, carried out by a consortium of researchers from eight European countries and Australia including the Department of Chemistry at the University of York, is unprecedented in its scope and has given scientists an unrivalled level of understanding of the way the organism can adapt to diverse conditions.

Billions of years of evolution have shaped the performance of B. subtilis cells and the research has provided novel insights into the regulatory processes that help them to maintain their metabolism in prime condition.

Published in two papers in the latest edition of Science, the findings will enable scientists to engineer B. subtilis to become an even more effective producer of metabolites for a wide range of industries from pharmaceutical and chemical manufacturing to the agri and food sectors. The work also has medical implications as it will help scientists to understand how bacteria deal with changing conditions during infection.

B. subtilis is able to survive and grow in diverse and changing environments. The research used expertise from different fields ranging from molecular biology to bioinformatics and mathematics to investigate the cell as a system of interacting molecular components and the strategies it uses to adapt to varying conditions.

The researchers acquired and analysed large experimental data sets which were used with mathematical models to capture the complexity of the cellular system. They analysed the genes expressed under more than 100 different conditions that mimic the natural and laboratory environment of the organism.

It was already known that the B. subtilis genome carries around 4,200 genes but the new research identified 512 new potential genes in the bacterium.

The project co-ordinator Dr Philippe Noirot, of the INRA Centre at Jouy-en-Josas, near Paris, says: "Besides their scientific novelty, these two studies also represent a potential blueprint for bacterial systems biology. Our work will potentially make B. subtilis an even more efficient producer of enzymes. The results and approaches used in our studies, suggest it is now possible to design specific experiments to unravel other, previously more intractable, cellular processes."

Professor Tony Wilkinson, of the York Structural Biology Laboratory, says: "The work has thrown up surprises. In one instance, where we expected that a few simple tweaks would be sufficient to achieve an adaptation, we observed wholesale changes involving almost half the genes in the organism."

Professor Uwe Sauer, of the Eidgenössische Technische Hochschule, Zürich, says: "The work represents a conceptual step forward in how to assess and understand cellular adaptation to new situations that is fundamental to basic science as well as applications in biotech and medical research."

Prof Jan Maarten van Dijl, of the University Medical Centre in Groningen, adds: "These studies help us to understand how bacteria deal with changing conditions during infection such as when normally commensal bacteria such as Staphylococcus aureus that live in the nose and throat adapt and invade the body and cause disease. This provides a foundation for research into the development of agents to combat these invasive bacteria."

More information: The papers ‘Global Network Reorganization During Dynamic Adaptations of Bacillus subtilis Metabolism’ and ‘Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis’ are published in the latest issue of Science.

Provided by University of York

Responding to the radiation threat

The New York Times recently reported that in the darkest moments of the triple meltdown last year of the Fukushima Daiichi nuclear power plant, Japanese officials considered the evacuation of the nearly 36 million residents of the Tokyo metropolitan area. The consideration of so drastic an action reflects the harsh fact that in the aftermath of a major radiation exposure event, such as a nuclear reactor accident or a "dirty bomb" terrorist attack, treatments for mass contamination are antiquated and very limited. The only chemical agent now available for decontamination – a compound known as DTPA - is a Cold War relic that must be administered intravenously and only partially removes some of the deadly actinides - the radioactive chemical elements spanning from actinium to lawrencium on the periodic table - that pose the greatest health threats.

Scientists at the U.S. Department of Energy (DOE)'s Lawrence Berkeley National Laboratory (Berkeley Lab) are developing a much more effective alternative that decontaminates a large number of the actinides likely to be part of the radiation exposure from a nuclear plant or weapon, including plutonium, americium, curium, uranium and neptunium. Furthermore, the Berkeley Lab treatment can be administered orally in the form of a pill, a necessity for prompt treatment in the event of mass contamination. Depending on the level of radiation exposure and how soon treatment can start, one of these pills would result in the excretion of approximately 90-percent of the actinide contaminants within 24 hours. Taking one pill daily for two weeks should be enough to remove virtually all of the actinide contaminants.

"With the expanding use of nuclear power and unfortunate possibility of nuclear weapon use, there is an urgent need to develop and implement an improved therapy for actinide contamination of a large population," says Rebecca Abergel, a chemist who leads the Bioactinide Group at Berkeley Lab's Glenn T. Seaborg Center. "We are now in the process of demonstrating that our actinide-specific decontaminating agents are ready for clinical development."

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Rebecca Abergel is the leader of the Bioactinide Group at Berkeley Lab's Glenn T. Seaborg Center, where a safe, effective radiation decontamination treatment is being developed. Credit: (Photo by Roy Kaltschmidt, Berkeley Lab)

Once actinides are ingested or inhaled, their radioactivity and cancerous interactions with cells and tissue demand they be immobilized and removed from the body as soon as possible. Abergel and her group are part of an effort at Berkeley Lab that began more than two decades ago under the leadership of Ken Raymond, a chemist who holds joint appointments with Berkeley Lab and the University of California (UC) Berkeley, where he is the Chancellor's Professor of Chemistry, in collaboration with the late Patricia Durbin. The primary goal of this project has been to identify sequestering agents that can encapsulate actinides into tightly bound cage-like chemical complexes for transport out of the body. The early focus of this research was on plutonium, the alpha particle-emitting actinide discovered by Berkeley Lab Nobel laureate Glenn Seaborg, and natural chelators, the crablike molecules that specifically bind with iron and other metal ions.

"Since the biochemical properties of plutonium(IV) and iron(III) are similar, we modeled our sequestering agents after the chelating unit found in siderophores," Raymond says. Siderophores are small molecules secreted by bacteria to extract and solubilize iron. "This biomimetic approach enabled us to design multidentate hydroxypyridonate ligands that are unrivaled in terms of actinide-affinity, selectivity and efficiency."

The two best candidate hydroxypyridonate ligands – nicknamed HOPO – developed by Abergel and her colleagues are a tetradentate, which has four chelating arms, and an octadentate, which has eight chelating arms. The "arms" in this case are atoms with pairs of electrons available for covalent bonding with an actinide.

"We've advanced our two candidate ligands through the initial phases of pre-clinical development by successfully scaling up synthesis to the 5-kilograms level and establishing baseline preparation and analytical methods suitable for manufacturing larger amounts under good manufacturing practice guidelines," Abergel says.

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Mass contamination from major radiation exposure events, such as the meltdown at Japan's Fukushima Daiichi nuclear power plant, require prompt treatment in the form of a pill, such as the treatment being developed at Berkeley Lab. Credit: (Satellite image from Digital Globe)

The team has also carried out extensive studies in animal models and human cell lines that established the two HOPO candidates as being highly effective and non-toxic at the tested doses. As for comparisons between the two, each has its own merits.

"A single octadentate HOPO can form a full actinide complex and results in more total actinide excretion," Abergel says. "However, it is easier for the smaller tetradentate HOPO to pass through biological membranes and access desired target sites in the body. Both warrant further development for emergency use in the case of a radiological event."

Abergel says the basic research and development phase of these two candidates has been completed and she and her group have started the process with the U.S. Food and Drug Administration (FDA) to determine what further data is needed to move into clinical trials. Typically at this stage of development a private pharmaceutical company would step in but it is difficult to attract private investors for a drug that will hopefully never be needed.

"As we move further along with the FDA process it should be easier to convince private pharmaceutical companies to get involved," Abergel says.

Provided by Lawrence Berkeley National Laboratory (news : web)

X-rays reveal how soil bacteria carry out surprising chemistry

Their result, reported today in Nature, details how five carbon atoms and one oxygen atom in the structure of lasalocid, a natural antibiotic produced by bacteria in soil (Streptomyces lasaliensis), can link into a six-membered ring through an energetically unfavorable chemical reaction. Unlocking this chemical pathway could enable scientists to synthesize many important chemicals currently found only in nature.

"Our study has a broad implication because the six-membered ring is a common structural feature found in hundreds of drug molecules produced by nature," said the study's principal investigator, Chu-Young Kim of the National University of Singapore. "We have actually analyzed the genes of six other organisms that produce similar drugs and we are now confident that the chemical mechanism we have uncovered applies to these other organisms as well."

According to "Baldwin's Rules for Ring Closure," which govern the way these rings form, this compound should contain a five-membered ring instead of the observed six-membered ring.

The solution to the molecular mystery depended in large part on a deeper understanding of the unique protein Lsd19, the catalyst that enables the formation of lasalocid's rings. To determine the protein's atomic structure, the researchers hit frozen crystals of Lsd19 with X-rays from SLAC's Stanford Synchrotron Radiation Lightsource and observed how the crystals diffracted the X-rays passing through. "You need atomic-level detail of the crystal's structure to understand what's really happening," said co-author Irimpan Mathews, a staff scientist at SLAC.

"The bugs have taught us a valuable chemistry lesson," Kim said. "With a new understanding of how nature synthesizes the six-membered rings, chemists may be able to develop novel methods that will enable us to produce these drugs with ease in the chemical laboratory. Alternatively, protein engineers may be able to use our results to develop a biofactory where these drugs are mass produced using a fermentation method. Either method will make more effective and more affordable drugs available to the public."

Kim's group has moved on to their next challenge: investigating how nature synthesizes the anti-cancer drug echinomycin. In the meantime, "The knowledge we have generated will help researchers in academia and industry to develop new methods for biological production of important polyether drugs," he said. "We are not talking about the distant future."

More information: Nature, DOI: 10.1038/nature10865

Provided by SLAC National Accelerator Laboratory (news : web)

The origin of organic magnets

 A theoretical model for the unusual occurrence of magnetism in organic molecules may help develop this class of material for electronics applications.

Electrical engineers are starting to consider materials made from organic molecules -- including those made from carbon atoms -- as an intriguing alternative to the silicon and metals used currently in electronic devices, since they are easier and cheaper to produce. A RIKEN-led research team has now demonstrated the origin of magnetism in organic molecules1, a property that is rarely found in this class of material, but is vital if a full range of organic electronic devices is to be created.

The permanent magnetic properties of materials such as iron stem from an intrinsic mechanism called ferromagnetism. Ferromagnetism in organic materials is rare because their atomic structure is fundamentally different from metals. One of the few examples identified to date is called TDAE-C60: a compound comprising spherical carbon cages attached to an organic molecule known as tetrakis-dimethylamino-ethylene. Since its identification in 1991, many theoretical and experimental studies have provided some insight into the mechanism driving this unexpected ferromagnetism, but the explanation was not definitive. A full understanding would help materials scientists to develop more advanced magnetic materials in the future. "A precise model for organic magnetism could aid the design of high-density recording materials for use in next-generation memories," says team member Hitoshi Yamaoka from the RIKEN SPring-8 Center, Harima.

Materials scientists are particularly interested in understanding the electronic structure of TDAE-C60 and how this relates to its ferromagnetic properties. To this end, Yamaoka and his colleagues from research institutes across Japan studied this material using a powerful technique known as photoelectron spectroscopy (PES). They fired x-rays at a single crystal of TDAE-C60, and this radiation excited electrons in the crystal, which then escaped from the surface. The researchers measured the number and the kinetic energy of these electrons from which they could infer information about the electronic structure.

"From these experiments on a single crystal we could establish an exact theoretical model for organic magnetism," explains Yamaoka. "We propose that the transfer of one electron from the TDAE to the C60 causes the magnetic properties of TDAE-C60." The existence of the resulting positively charge TDAE state was also supported by the team's theoretical calculations.

With this thorough understanding of organic magnetism, the next step will be to apply the material to practical applications. "The problem with the TDAE-C60 organic magnet, however, is that the magnetism only appears at temperatures below 16 kelvin," says Yamaoka. "The next step will be to raise this transition point."

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