WASHINGTON, DC—November 4, 2014—An international team of bioengineers has boosted the ability of bacteria to produce isopentenol, a compound with desirable gasoline properties. The finding, published in mBio®, the online open-access journal of the American Society for Microbiology, is a significant step toward developing a bacterial strain that can yield industrial quantities of renewable bio-gasoline.
The metabolic engineering steps to produce short-chain alcohol solvents like isopentenol in the laboratory bacteria Escherichia coli have been worked on extensively by many research groups, explains Aindrila Mukhopadhyay, director of host engineering at the Joint BioEnergy Institute in Emeryville, California and senior author on the study.
“Biofuels are one tool in the array of alternative energy solutions that can be used in our infrastructure immediately,” she says. Sustainably produced fuel compounds can be added directly into gasoline blends used today to offset reliance on fossil fuels and also lower the net carbon emissions from vehicles.
“But the solvent-like compounds inhibit microbial growth and that was an aspect that we realized would come up sooner rather than later,” says Mukhopadhyay, who holds a joint appointment at Lawrence Berkeley National Laboratory. “We wanted to look at that aspect with a systems biology approach — could we engineer bacteria to also tolerate the solvent it is producing?”
Improving tolerance is key to moving production toward levels that are industrially relevant. Industrial production requires a robust strain that can stably produce for longer periods of time and withstand the accumulation of the solvent-like biofuel.
To address this challenge, the team, which also included researchers from Nanyang Technological University in Singapore, National University of Singapore, and the University of California, Berkeley, treated a non-producing E. coli strain with isopentenol by adding it to the culture. As the bacteria responded to the solvent-stressor, the team measured which genes were shifted up or down by looking at messenger RNA transcripts across the entire genome.
They chose 40 genes that the bacteria cranked up in response to isopentenol—presumably because their actions helped mitigate the toxicity in some way. Next, they overexpressed each one in a bacterial strain actively producing isopentenol to see which ones might improve the strain’s growth.
Of the eight genes that rescued growth, two stood out as promising—MetR, a biosynthesis regulator, that improved isopentenol production by 55%, and MdlB, a transporter, that improved production by 12%. If the researchers bumped up the levels of the MdlB transporter protein inside the cells even further, they saw production improve by as much as 60% over the original strain.
“Finding a transporter really appealed to us because it has the potential to export the final solvent product out of the cell,” says Mukhopadhyay. “And in this case, once enough alcohol gets outside the cell, it might phase separate and not even be accessible to the organism anymore.” In other words, the biofuel would separate away to sit atop the watery broth the bacteria live in.
As an added bonus, the MdlB protein is a good candidate for directed evolution experiments that could improve the performance and specificity of the transporter for shuttling isopentenol out as quickly as possible. Combining a more efficient transporter with other genes that improved tolerance might produce a strain that can generate bio-gasoline for the gas pump in the near future.
This research was supported by the U.S. Department of Energy and the National Research Foundation of Singapore. The article can be found online at http://mbio.asm.org/content/5/6/e01932-14.
WASHINGTON, DC – November 4, 2014 – Filoviruses like Ebola “edit” genetic material as they invade their hosts, according to a study published this week in mBio®, the online open-access journal of the American Society for Microbiology. The work, by researchers at the Icahn School of Medicine at Mount Sinai, the Galveston National Laboratory, and the J. Craig Venter Institute, could lead to a better understanding of these viruses, paving the way for new treatments down the road.
Using a laboratory technique called deep sequencing, investigators set out to investigate filovirus replication and transcription, processes involved in the virus life cycle. They studied the same Ebola virus species currently responsible for an outbreak in West Africa, and also analyzed a related filovirus, Marburg virus, that caused a large outbreak in Angola in 2005 and recently emerged in Uganda. The scientists infected both a monkey and human cell line with both viruses, and analyzed the genetic material produced by each virus, called RNA.
Their results highlight regions in Ebola and Marburg virus RNAs where the polymerase of the virus (a protein that synthesizes the viral RNA) stutters at specific locations, adding extra nucleotides (molecules that form the building blocks of genetic material like DNA and RNA), thereby “editing” the new RNAs. The study found new features at a described RNA editing site in the Ebola glycoprotein RNA, which makes the protein that coats the surface of the virus. Their work also identified less frequent but similar types of editing events in other Ebola and Marburg virus genes – the first time this has been demonstrated.
Because of these messenger RNA modifications, Ebola and Marburg are potentially making proteins “that we previously didn’t realize,” said Christopher F. Basler, PhD, senior study author and professor of microbiology at Mount Sinai.
“The bottom line is we know these changes occur but we don’t yet know what it really means in the biology of the virus,” Basler said. There are many aspects of how the viruses replicate that aren’t yet understood, he said, “so we need a complete description of how they grow to develop new strategies used to treat the infections.”
The study also illustrated how the filoviruses express their genes, and deep sequencing identified all seven messenger RNAs within six hours of infection.
“Our study suggests that the Ebola virus is making forms of proteins previously undescribed,” said lead author Reed Shabman, PhD, an assistant professor at the J. Craig Venter Institute in Rockville, Md. Shabman was at Mount Sinai when the study was initiated. “Understanding the products of these viruses is critical to understanding how to target them.”
In addition, he said, proteins produced by the glycoprotein editing site are associated with virulence in animals, “so it’s of great interest to understand how that protein is made, and in as much detail as possible.”
“We infer that this probably contributes to how the virus grows in a person or an animal,” Basler said.
Further study is needed to determine the biological significance of these findings and how these processes are regulated, Basler said.
The study was supported by the National Institutes of Health and the J. Craig Venter Institute. The article can be accessed freely online at http://mbio.asm.org/content/5/6/e02011-14.
WASHINGTON, DC – September 30, 2014 – A species of gut bacteria called Clostridium ramosum, coupled with a high-fat diet, may cause animals to gain weight. The work is published this week in mBio®, the online open-access journal of the American Society for Microbiology.
A research team from the German Institute of Human Nutrition Potsdam-Rehbruecke in Nuthetal observed that mice harboring human gut bacteria including C. ramosum gained weight when fed a high-fat diet. Mice that did not have C. ramosum were less obese even when consuming a high-fat diet, and mice that had C. ramosum but consumed a low-fat diet also stayed lean.
Previous studies have found C. ramosum and other representatives of the Erysipelotrichi class in obese humans, said senior study author Michael Blaut, PhD, head of the institute’s Department of Gastrointestinal Microbiology. This suggests that growth of this organism in the digestive tract is stimulated by high-fat diets, which in turn improves nutrient uptake and enhances the effect of such diets on body weight and body fat.
“We were surprised that presence or absence of one species in a defined bacterial community affected body weight and body fat development in mice,” says Blaut.
Blaut and colleagues investigated the role of C. ramosum in three groups of mice: some harbored a simplified human intestinal microbiota (bacteria) of eight bacterial species including C. ramosum; some had simplified human intestinal microbiota except for C. ramosum, and some had C. ramosum only. The researchers called the first group SIHUMI, the second group SIHUMIw/oCra and the third group Cra. Mice were fed either a high-fat diet or low-fat diet for four weeks.
After four weeks eating a high-fat diet, the mouse groups did not differ in energy intake, diet digestibility, and selected markers of low-grade inflammation. However, SIHUMI mice and Cra mice fed a high-fat diet gained significantly more body weight and body fat, which implies that they converted food more efficiently to energy than did the SIHUMIw/oCra mice. By contrast, all groups of mice fed a low-fat diet stayed lean, indicating that the obesity effect of C. ramosum only occurred on high-fat diets.
The obese SIHUMI and Cra mice also had higher gene expression of glucose transporter 2 (Glut2), a protein that enables absorption of glucose and fructose, and fat transport proteins including fatty acid translocase (Cd36).
“Our results indicate that Clostridium ramosum improves nutrient uptake in the small intestine and thereby promotes obesity,” Blaut said. Associations between obesity and increased levels of lipopolysaccharides (components of the cell wall of gram-negative bacteria) causing inflammation, or increased formation of molecules called short chain fatty acids, reported by other researchers, were not found in this study, he said: “This possibly means that there is more than one mechanism underlying the promotion of obesity by intestinal bacteria.”
Through additional studies Blaut said he hopes to learn more about how C. ramosum affects its host’s energy metabolism and whether similar results occur in conventional mice given the bacteria. “Unraveling the underlying mechanism may help to develop new strategies in the prevention or treatment of obesity,” he said.
The current study was supported by the German Institute of Human Nutrition Potsdam-Rehbruecke.
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mBio® is an open access online journal published by the American Society for Microbiology to make microbiology research broadly accessible. The focus of the journal is on rapid publication of cutting-edge research spanning the entire spectrum of microbiology and related fields. It can be found online at http://mbio.asm.org.
The American Society for Microbiology is the largest single life science society, composed of over 39,000 scientists and health professionals. ASM's mission is to advance the microbiological sciences as a vehicle for understanding life processes and to apply and communicate this knowledge for the improvement of health and environmental and economic well-being worldwide.
WASHINGTON, DC – October 20, 2014 (updated February 2, 2015) -- Researchers found that Lactobacillus species, a bacteria commonly seen in yogurt cultures, when present in the guts of mice, mitigate lupus symptoms, while Lachnospiraceae, a type of Clostridia, correlate with the worsening of these symptoms. This research was published ahead of print in the journal Applied and Environmental Microbiology.
In the study, investigators found that mice with lupus had higher levels of Lachnospiraceae (a type of Clostridia), and lower levels of Lactobacillus compared to mice in the control group. They also compared male and female mice, and found that these differences were present only in females.
They also monitored the gut microbiota over time in both the lupus and control mice, and found that in the former, Clostridia increased in both early and late stages of the disease.
"Our results suggest that the same investigation should be performed in human subjects with lupus," says principal investigator Xin Luo of Virginia Tech, Blacksburg, VA.
In further experiments, the investigators treated the symptoms in the lupus mice with either retinoic acid alone or vitamin A plus retinoic acid. The latter worsened the symptoms—surprisingly, Luo says, because it had been expected to reduce them—and in those mice, Clostridia increased, while Lactobacillus declined. Retinoic acid alone improved the symptoms, with opposite population changes in the gut bacteria.
"We were inspired in part to perform this research by a study on type 1 diabetes, which found that that disease is dependent on gut microbiota," says Zhang. "Like type 1 diabetes, lupus is an autoimmune disease that is even more prevalent [than type 1 diabetes] in women."
Applied and Environmental Microbiology is a publication of the American Society for Microbiology (ASM). The ASM is the largest single life science society, composed of over 39,000 scientists and health professionals. Its mission is to advance the microbiological sciences as a vehicle for understanding life processes and to apply and communicate this knowledge for the improvement of health and environmental and economic well-being worldwide.
WASHINGTON, DC – September 29, 2014 -- Plants have a symbiotic relationship with certain bacteria. These ‘commensal’ bacteria help the pants extract nutrients and defend against invaders – an important step in preventing pathogens from contaminating fruits and vegetables. Now, scientists have discovered that plants may package their commensal bacteria inside of seeds; thus ensuring that sprouting plants are colonized from the beginning. The researchers, from the University of Notre Dame, presented their findings today at the 5th ASM Conference on Beneficial Microbes.