Synchrotron Infrared Unveils a Mysterious Microbial Community

Berkeley Lab scientists join an international collaboration to understand how archaea and bacteria work together deep in a cold sulfur spring
Paul Preuss 510-486-6249 paul_preuss@lbl.gov

http://newscenter.lbl.gov/feature-stories/2013/01/22/sir-archaea/?utm_source=Synchrotron+Infrared+Unveils+a+Mysterious+Microbial+Community&utm_campaign=Mysterious+Microbial+Community&utm_medium=email
In the fall of 2010, Hoi-Ying Holman of the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) was approached by an international team researching a mysterious microbial community discovered deep in cold sulfur springs in southern Germany.
“They told me what they were doing and said, ‘We know what you contributed to the oil-spill research,’” recalls Holman, who heads the Chemical Ecology group in Berkeley Lab’s Earth Sciences Division. “They wondered if I could help them determine the biochemistry of their microbe samples.”
Holman had co-authored a report in Science about bacteria in the Gulf of Mexico that thrived on the Deepwater Horizon oil plume. Using infrared spectromicroscopy at the Berkeley Synchrotron Infrared Structural Biology (BSISB) facility, which she directs at the Advanced Light Source (ALS), Holman helped determine how the novel bug obtained energy by eating the spilled crude. No stranger to subsurface bioscience, Holman would soon add a new actor to her cast of remarkable microbes.
Not extreme, but weird anyway
The name Archaea means “ancient things,” but Archaea were recognized as a distinct domain of life less than forty years ago. First thought to be exclusively extremophiles – lovers of boiling hot springs, deep-sea black smokers, acid mine runoff, and other inhospitable environments – more and more archaea are found thriving in moderate and cold environments, almost always as minority members of much larger microbial communities.
A unique exception to this pattern was discovered less than 10 years ago in the Sippenauer Moor in Germany. In microbial mats in this cold sulfur spring’s outflow, the SM1 Euryarchaeon lives in roughly equal abundance with bacteria in a community that forms symbiotic “strings of pearls”: the archaea fill the “pearls” and filamentous bacteria cover the pearl surfaces and form strings between them. The two kinds of microbes were assumed to be syntrophic – dependent on each other for nourishment – but the biochemical details were a mystery.
Christine Moissl-Eichinger of the University of Regensburg was among the SM1 Euryarchaeon’s discoverers. Before long what she calls “another amazing lifestyle” of the new archaeon emerged; biofilms that grew deep below the surface of another cold sulfur spring, the nearby Muehlbacher Schwefelquelle. Moissl-Eichinger and her team collected samples of the slime-like biofilm – which first seemed to be pure SM1 – on net traps underwater.

Synchrotron infrared biology
Microbial communities are essential to cleaning up subsurface pollution – including residues of metals and radionuclides at sites once involved in nuclear weapons research and assembly. Susan Hubbard heads the Earth Sciences Division’s (ESD’s) Environmental Remediation and Water Resources Program, with several projects that take advantage of synchrotron radiation Fourier-transform infrared spectromicroscopy – SR-FTIR for short – to understand biochemical changes in living microorganisms during the oxidation or reduction of uranium and chromium wastes.
SR-FTIR doesn’t require intrusive cell labeling, it can readily and nondestructively distinguish archaea from bacteria and assess their biochemistry, and it can follow changes in chemical composition of different members of the same microbial community over time. A mainstay of the Berkeley Synchrotron Infrared Structural Biology program (BSISB), the technology is increasingly recognized as an indispensable tool for microbiological investigation.
Microbial ecologist Eoin Brodie of ESD uses SR FTIR to understand how communities of soil microbes change as the climate changes, and how the composition of wood materials changes as they pass through a beetle’s gut. Meanwhile a team from halfway around the world is investigating how climate change affects crop growth in Australia, examining the “rizosphere,” the subsurface realm where symbiotic microorganisms extend from plant roots to collect water and nutrients such as nitrogen.
“At the BSISB we examine living root hairs in our microfluidic chamber, with just enough moisture to sustain life but not enough to absorb the infrared beam,” says its director Hoi-Ying Holman. “We can see how root growth is affected by introducing different microorganisms and watching the microbes and root hairs interact in real time.”
Examining life’s changing chemistry in real time isn’t confined to microorganisms. One example is wound healing, a research project by a team from the University of California at Davis and the University of Aberdeen in Scotland. Living human cells are imaged under the infrared beam to see what molecular changes are triggered in response to electric fields.
BSISB’s beamline 5.4 is unique at the Advanced Light Source. “Unlike x-rays, long-wavelength infrared reflects from ordinary mirrors and can be steered around sharp corners,” explains ALS infrared expert Michael Martin. “So instead of the crowded ALS floor, where x-ray beamlines are packed side by side, BSISB can spread out on its concrete-block roof.”
Extra room on the roof means live samples can be prepared in situ and studied with an array of imaging and spectroscopic techniques—an ideal facility for life sciences, environmental sciences, materials sciences, and a range of applications limited only by the users’ imaginations.

To augment their already extensive research, Moissl-Eichinger and Alexander Probst of her staff brought the Regensburg samples to Berkeley Lab, initially attracted by the PhyloChip, a DNA microarray invented by Berkeley Lab’s Gary Andersen and Todd DeSantis and their colleagues. Because the PhyloChip probes for the 16S rRNA gene, found in all Bacteria and Archaea, it can quickly and accurately sort through all known species in a sample – including those, like SM1 and many other microorganisms, that can’t be grown in culture.
Probst and DeSantis, both now with Second Genome, Inc., and Andersen were joined by Kasthuri Venkateswaran of the Jet Propulsion Laboratory, a member of NASA’s Biotechnology and Planetary Protection Group. Probst wanted to know who was living where in the subsurface sulfur-spring samples; Venkateswaran’s interest is understanding the role of Archaea in space and analogous sites. Although SM1 was by far the dominant species in the subsurface community, they found that small amounts of other archaea were present as well – and about five percent of the community consisted of bacteria.
Bring on the synchrotron
Led by Andersen, the PhyloChip’s inventors had contributed to the oil-spill research, and their previous association with Holman brought her and her BSISB colleagues aboard the SM1 research team.
“Lots of biochemical techniques can tell you what’s in a sample – lipids and carbohydrates, for example – but just because they’re there doesn’t mean they interact,” says Holman’s colleague Giovanni Birarda, a member of the BSISB staff. “Synchrotron radiation–based Fourier-transform infrared spectromicroscopy – SR-FTIR – takes images and spectra of the same sample, so you can map the chemical relationships by combining the images with spectra that identify where the archaea and bacteria are.”
Holman says, “The main difference is in their membrane lipids. Bacterial membrane lipids consist of fatty acids with long alkylic chains” – functional groups of singly bonded carbon and hydrogen atoms – “which have only one to two terminal methyl groups,” groups with one carbon and three hydrogen. “By contrast, archaeal membrane lipids generally consist of branched and saturated isoprenes” – a more complex common hydrocarbon – “and are relatively less alkylic but have more methyl groups.”
By revealing the bright spectral signals of alkylic and methyl groups, together with sulfur functional groups, synchrotron FTIR unambiguously identified the sulfate-reducing metabolic activity of the bacteria within the SM1 samples. The archaeal cells themselves showed no such activity, leading the researchers to posit a thriving mutual metabolism of the archaea and bacteria.
In many cases, such syntrophy requires close physical association. Covering the surface of each SM1 cell the researchers found spines made of three protein strands, equipped with terminal hooks where the strands divided. Moissl-Eichinger named them hami, Latin for barbs or hooks. These “nano-grappling hooks” apparently hold the microbial partners together, working in synchronization. The major hami protein is unlike any known proteinaceous archaeal or bacterial filaments.
How SM1 Euryarchaea interact with their bacterial partners may be a model for understanding other syntrophic relations essential to the carbon and sulfur cycles on which Earth’s life depends. So far found in just two sites in Germany, the species is the only example yet of an archaeon that dominates a biological ecosystem – but related species have been found in sulfur springs as far afield as Turkey and may be widespread.
DOE’s Office of Science supported building and equipping the BSISB and also supports the ALS

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“Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm,” by Alexander J. Probst, Hoi-Ying N. Holman, Todd Z. DeSantis, Gary L. Andersen, Giovanni Birarda, Hans A. Bechtel, Yvette M. Piceno, Maria Sonnleitner, Kasthuri Venkateswaran, and Christine Moissl-Eichinger, appears in advanced online publication of The ISME Journal, 22 November 2012, and is available at http://www.nature.com/ismej/journal/vaop/ncurrent/abs/ismej2012133a.html.
The Science article on novel hydrocarbon-degrading bacteria associated with the Deepwater Horizon oil spill may be found at http://www.sciencemag.org/content/330/6001/204.abstract.
More about the Berkeley Synchrotron Infrared Structural Biology Program is at http://infrared.als.lbl.gov/content/structuralbiology/overview.
For Susan Hubbard’s research, including bioremediation of uranium-contaminated sites, see http://esd.lbl.gov/about/staff/susanhubbard/ and http://esd.lbl.gov/research/programs/erwr/
Eoin Brodie’s microbial research is described at http://envmicro.wordpress.com/
More about how climate change may affect microbial communities important to crop growth in Australia is at http://www.csiro.au/en/Organisation-Structure/Divisions/Plant-Industry/michellewatt.aspx
Lawrence Berkeley National Laboratory addresses the world’s most urgent scientific challenges by advancing sustainable energy, protecting human health, creating new materials, and revealing the origin and fate of the universe. Founded in 1931, Berkeley Lab’s scientific expertise has been recognized with 13 Nobel prizes. The University of California manages Berkeley Lab for the U.S. Department of Energy’s Office of Science. For more, visit www.lbl.gov.
DOE’s Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit the Office of Science website at science.energy.gov/.

Molecular Spectroscopy Tracks Living Mammalian Cells in Real Time as They Differentiate

(Berkeley Lab scientists observed phosphorylation in living PC12 cells stimulated by nerve growth factor as they differentiated and sent out neuron-like neurites. The researchers imaged individual cells and simultaneously obtained absorption spectra using synchrotron radiation from the Advanced Light Source. Cells not stimulated with nerve growth factor did not differentiate and showed different infrared absorption spectra)   http://newscenter.lbl.gov/feature-stories/2012/04/30/phosphorylation/

Berkeley Lab scientists demonstrate the promise of synchrotron infrared spectroscopy of living cells for medical applications

April 30, 2012

Knowing how a living cell works means knowing how the chemistry inside the cell changes as the functions of the cell change. Protein phosphorylation, for example, controls everything from cell proliferation to differentiation to metabolism to signaling, and even programmed cell death (apoptosis), in cells from bacteria to humans. It’s a chemical process that has long been intensively studied, not least in hopes of treating or eliminating a wide range of diseases. But until now the close-up view – watching phosphorylation work at the molecular level as individual cells change over time – has been impossible without damaging the cells or interfering with the very processes that are being examined.
“To look into phosphorylation, researchers have labeled specific phosphorylated proteins with antibodies that carry fluorescent dyes,” says Hoi-Ying Holman of the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab). “That gives you a great image, but you have to know exactly what to label before you can even begin.”
Holman and her coworkers worked with colleagues from the San Diego and Berkeley campuses of the University of California to develop a new technique for monitoring protein phosphorylation inside single living cells, tracking them over a week’s time as they underwent a series of major changes.
“Now we can follow cellular chemical changes without preconceived notions of what they might be,” says Holman, a pioneer in infrared (IR) studies of living cells who is director of the Berkeley Synchrotron Infrared Structural Biology program at Berkeley Lab’s Advanced Light Source (ALS) and head of the Chemical Ecology Research group in the Earth Sciences Division . “We’ve monitored unlabeled living cells by studying the nonperturbing absorption of a wide spectrum of bright synchrotron infrared radiation from the ALS.”
The researchers report their results in the American Chemical Society journal Analytical Chemistry.
Phosphorylation fundamentals
Phosphorylating enzymes add one or more phosphate groups to three amino-acid residues common in proteins – serine, threonine, or tyrosine – which activates the proteins; removing the phosphate reverses the process. The research goal is to learn exactly when proteins such as enzymes and receptors are switched on and off by phosphorylation, and which cells within a population are responding to cause specific changes – for example, during differentiation of a progenitor cell into its functional form.
To avoid killing cells or introducing modified proteins or foreign bodies that may alter their behavior, scientists can use a method called Fourier-transform infrared (FTIR) spectromicroscopy; because infrared light has lower photon energy than x-rays, it can peer inside living cells without damaging them. Different components and different states of the cell absorb different wavelengths of the broad infrared spectrum; applying the Fourier-transform algorithm allows signals of all frequencies to be recorded simultaneously, pinpointing when, where, and what chemical changes are occurring.
Most infrared sources are dim, however, so the information from typical IR set-ups is limited in resolution and has a low signal-to-noise ratio. Infrared from the ALS’s synchrotron light source is a hundred to a thousand times brighter.

PC12 cells treated with nerve growth factor underwent a series of changes due to phosphorylation. Beginning at Day 3 they sent out neurites, resembling the growth of nerve cells. Spectromicroscopy at beamline 1.4.3 of the Advanced Light Source tracked specific local chemical changes in the living cells.

Previously Holman and her colleagues have used IR beamline 1.4.3, managed by Berkeley Lab’s Michael Martin and Hans Bechtel, to obtain spectra from living organisms in rock, soil, and water. They have monitored ongoing biochemistry within living bacteria adapting to stress, and more recently within individual skin connective tissue cells (fibroblasts) from patients with mitochondrial disorders. (Mitochondria are the cellular organelles commonly known as the “power-plants” of the cell.)
The present study was done with a line of cultured cells called PC12. When nerve growth factor, a small protein, is introduced into a PC12 cell, the cell begins to send out neurites resembling the projections from nerve cell bodies. Although originally derived from a tumor of the rat’s adrenal gland, PC12 has become, rather counterintuitively, a valuable model of how nerve cells differentiate from their unspecialized progenitors.
Berkeley Lab postdoctoral fellow Liang Chen began the current experiments by introducing nerve growth factor to groups of PC12 cells to induce them to differentiate; one group of cells was left untreated as a control. The cells were cultured on gold-coated slides in chambers maintained at body temperature in a humidified environment and supplied with nutrients. Individual cells of a group were positioned under the infrared beam at the beamline 1.4.3 endstation.
FTIR spectra were collected before and after the nerve growth factor was introduced. After stimulation, the spectra were taken first at short intervals, from two to sixty minutes apart. Additional spectra were collected of cells in other groups on the third, fifth, and seventh day of continued stimulation.
The first day’s spectra revealed spikes in phosphorylation activity within minutes after the addition of the nerve growth factor, in concert with changes in the ratios of such important chemical contents of the cell as proteins, carbohydrates, and lipids. Phosphorylation subsequently waned, then picked up again in another burst of activity on Day 3, just as the cells began to extend neurites.
By comparing results with quantum chemistry simulations by Berkeley Lab’s Zhao Hao — predicting what should be observed from first principles — as well as with results from partial studies using other methods, the researchers confirmed the monitoring of phosphorylation phases, their timing, and their target proteins, along with associated changes in other substances in the cell.

In the top panel, different modes of imaging of the same cell show the differences between visible light microscopy and fluorescence imaging, and in the lower panel, the images resulting from Fourier-transform infrared spectromicroscopy. Infrared absorption at different frequencies pinpoints different cell components at specific locations in the living cell. 

A new technique takes off
“This experiment was a proof of the concept,” says Liang Chen. “We demonstrated the dynamics of protein phosphorylation in controlling differentiation in this biological system using synchrotron infrared spectromicroscopy, and we pointed the way to answering the many questions a biologist has to ask about measuring the coordination of specific processes in real time.”
Although in this first experiment the team was not able to follow individual cells continuously, they were able to monitor differentiation in groups of cultured PC12 cells in real time, without labeling or any other invasive procedure. It was the first step in an ambitious inquiry into the real-time biochemistry of living mammalian cells over the long term.
At beamline 1.4.3., with the help of new team members Kevin Loutherback and Rafael Gomez-Sjoberg, the team is designing equipment to maintain mammalian cells in a thin layer of culture media that will keep them healthy yet not interfere with the infrared beam, while automatically monitoring and adjusting temperature, humidity, and nutrient ratios, and removing waste products. This will allow data on individual cells to be gathered continuously throughout the entire phosphorylation process.
Meanwhile the Berkeley Synchrotron Infrared Structural Biology program at ALS beamline 5.4 is building multimodal facilities that will monitor cell development in human cells, bacteria, and plants, within soils, minerals, and other environments, via “hyperspectromicroscopy” – from the ultraviolet through visible light and deep into the infrared. Researchers will be able to choose the frequency window (or combination of windows) best suited to the sample and the conditions – in Holman’s words, “to watch almost everything at once.”
Says Holman, “Many researchers from the medical communities are interested in using the technology, and we are particularly interested in collaborating with university centers and private firms that are seeking a broad view of how promising drugs act within specific cells.”
Some of the projects will target Alzheimer’s disease, macular degeneration of the retina in diabetes, and mitochondrial diseases in children. In addition, specific processes like protein glycation can also be identified. Since different cells and different organisms respond differently, the eventual goal is to develop specific ways to screen the mechanisms of individual medicines.

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“Synchrotron infrared measurements of protein phosphorylation in living single PC12 cells during neuronal differentiation,” by Chen et al, appears in Analytical Chemistry, online at http://pubs.acs.org/doi/full/10.1021/ac300308x.
The research was performed at the Advanced Light Source and the Berkeley Synchrotron Infrared Structural Biology Program, which are supported by the U.S. Department of Energy Office of Science.
Learn more about infrared beamlines at the Advanced Light Source at infrared.als.lbl.gov/.
The Advanced Light Source is a third-generation synchrotron light source producing light in the x-ray region of the spectrum that is a billion times brighter than the sun. A DOE national user facility, the ALS attracts scientists from around the world and supports its users in doing outstanding science in a safe environment. For more information visit www-als.lbl.gov/.
DOE’s Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit the Office of Science website at science.energy.gov/.
Lawrence Berkeley National Laboratory addresses the world’s most urgent scientific challenges by advancing sustainable energy, protecting human health, creating new materials, and revealing the origin and fate of the universe. Founded in 1931, Berkeley Lab’s scientific expertise has been recognized with 13 Nobel prizes. The University of California manages Berkeley Lab for the U.S. Department of Energy’s Office of Science. For more, visit www.lbl.gov.