MICROBIOLOGY

Mass Spec Measures Nitrogen Fixed By Bacteria

Metabolism of Element Imaged in Shipworms

Long before humans ever set sail, wooden rafts ferried passengers over rivers, lakes, and oceans. Fallen trees and branches have provided makeshift vessels for countless plants and animals. Indeed, many species owe their evolutionary expansion to a floating tree trunk or limb. It might stand to reason that some enterprising animals would make a meal of the water-borne lumber. Yet almost none do, and for good reason. Wood is notoriously difficult to digest and completely lacking in protein and many other nutrients.

Graham Ramsay

Multi-isotope imaging mass spectrometry (MIMS), which can detect isotope-containing compounds, “is basically applicable to all kinds of research,” said Claude LeChene (right), with Greg McMahon. LeChene is collaborating with numerous Harvard groups on diverse projects such as heart regeneration, tracking stem cells, and transplant regeneration.

“It’s junk food,” said Daniel Distel, director of the Ocean Genome Legacy (OGL). One animal that breaks this rule is the shipworm, which feasts entirely on wood. Seventeenth- and 18th-century naturalists marveled at how the organism—which looks like a worm but is actually a mollusk—uses its twin shells to bore into wood (sailors dreaded the way it ate ships, docks, and pilings).

Twenty years ago, researchers discovered that the creature owes its wood-eating ability to an ingenious species of bacteria living in its gills. Teredinibacter turnerae, which are packed by the hundreds into cells called bacteriocytes, secrete two enzymes. One digests wood and another grabs nitrogen from the atmosphere and converts it into ammonia and other organic compounds, which can be used to produce protein. But because the bacteria are so minute, no one had actually caught an individual T. turnerae in the act of grabbing and fixing nitrogen.

HMS researcher Claude LeChene, director of the National Resource for Imaging Mass Spectrometry at Brigham and Women’s Hospital, working with Distel and colleagues, has done just that. In a striking series of images, rendered in multiple colors, the researchers have caught not just individual bacteria in the act of fixing nitrogen, but have also shown the nitrogen-containing organic compounds as they are sent from the gill to other parts of the shipworm’s body.

Using a method developed by LeChene—essentially brushing atoms off the surface of cells at predetermined locations and then counting the number of dislodged nitrogen atoms at those spots—they were able to identify how much nitrogen one bacterium fixes as opposed to another.

Shipshape. The shipworm (left) captures and fixes nitrogen by means of bacteria housed in cells located in its gills. The nitrogen is then released and sent to other parts of the body. The flow of nitrogen from the gills to the periphery is graphically rendered through a mass spectrometry approach called MIMS (below), with the brightest magenta representing areas of highest nitrogen uptake.

At right, MIMS shows individual bacteria located within the gill cells in the act of fixing nitrogen. (Also see video.)

Until the current study, which appears in the Sept. 14 Science, no one had seen any microorganism, including the ubiquitous nitrogen-fixing bacteria that shroud the roots of plants—and ultimately provide humans with the vital element—carry out the life-supporting function.

“Microbiologists, because their organisms are so tiny, cannot measure physiology in a single organism. Now, suddenly, this method provides a way to measure physiological processes in a single bacterial cell. That’s revolutionary,” said OGL’s Distel, whose organization is dedicated to gathering the genomes of endangered marine species.

According to Marcel Kuypers of the Max Planck Institute for Marine Microbiology, who wrote a perspective accompanying the new study, LeChene’s method, multi-isotope imaging mass spectrometry (MIMS), “is poised to reveal the metabolic diversity of the planet’s microorganisms, 99 percent of which have eluded cultivation.”

Nitrogen Tracer
The idea of using nitrogen to study metabolic processes goes back decades to the brilliant but ill-fated German Jewish scientist Rudolp Schoenheimer. Having fled Nazi Germany in 1933, Schoenheimer came to New York City, where he began working with Harold Urey, the Nobel Prize–winning chemist. Urey had isolated the nitrogen isotope N15. Schoenheimer fed mice amino acids laced with N15 and, using a mass spectrometer he built, observed that the isotope was incorporated into the animals’ organs to make proteins. “He literally discovered that there was a rapid turnover of proteins in the cells of the body,” said LeChene. Schoenheimer gave the 1941 Dunham Lectures at HMS but did not live to see their publication as a book, The Dynamical State of Bodily Constituents, the following year. Plagued by depression and the worsening situation in Europe, Schoenheimer committed suicide at the age of 43.

Soon after, researchers began harnessing the radioactive properties of C14, tritium (H3), and other compounds to label metabolic processes, leaving nitrogen, an extremely stable element, behind. Carbon and hydrogen are prevalent in carbohydrates and lipids as well as proteins, while nitrogen is found only in proteins and nucleic acids. Convinced that the protein-specific nitrogen would make a superior label for studying metabolic processes, LeChene began looking for a way to image and quantify the element. “I wanted to take up from where Schoenheimer left off, to basically do the same studies but at the cellular and intracellular level,” he said.

SIMS to MIMS
In the 1990s, LeChene collaborated with the French physicist Georges Slodzian, who had invented a method, secondary ion mass spectrometry (SIMS), for imaging compounds. In classical mass spectrometry, a compound is vaporized, producing ions that are then separated by mass and charge. In SIMS, the sample is not vaporized but, instead, is bombarded by a beam of charged particles, or primary ions. This causes a small cloud, or sputter, of atoms to be dislodged from the surface, some of which are ionized. These secondary ions are then sent into the mass spectrometer, where they are analyzed.

To adapt the method for pinpointing and measuring nitrogen required making significant modifications, essentially resulting in a new type of SIMS instrument (see sidebar). Over the past few years LeChene has been perfecting the new MIMS approach. Meanwhile, Distel had his own reasons for wanting to image nitrogen. For years, he had been fascinated by T. turnerae and its uncanny ability to fix nitrogen, but had become frustrated by the methods available, which consisted largely of exposing the bacteria to nitrogen, “grinding them up, and measuring the amount of nitrogen incorporated into the gemisch,” he said. He got wind of LeChene’s efforts and approached him with the idea of imaging individual T. turnerae in the act of fixing nitrogen.

After a promising initial test, LeChene and Distel, along with Greg McMahon, HMS instructor in medicine at BWH, and Yvette Luyten, graduate assistant at OGL, conducted a series of experiments. They cultured T. turnerae in the presence of N15 gas and did the same with a non–nitrogen-fixing species of bacteria, Enterococcus faecalis. They mixed the two species and put them on a slide, subjecting them to MIMS analysis. The T. turnerae cells were full of nitrogen. As for E. faecalis, “it didn’t incorporate one zilch of N15,” said LeChene.

Next, the researchers placed whole shipworms, burrowed into pieces of wood, in cylinders filled with N15. The animals stayed there for eight days, after which they were preserved, embedded in epoxy, and thin-sectioned. The sections were analyzed, first by electron microscopy to identify the bacteria-filled gill cells. The same slides were then subjected to MIMS analysis. The method picked out the nitrogen-filled bacteriocytes and even individual T. turnerae. Intriguingly, significant amounts of nitrogen were detected at the periphery of the shipworm body, where there are no T. turnerae, suggesting that the nitrogen produced by the bacteria was actually being used by the shipworm.

Though idiosyncratic, the shipworm results could apply more generally to the more familiar soil-dwelling nitrogen-fixing bacteria. “We’ll probably learn about the process of nitrogen fixation in general, which could be critical to our understanding of food production,” said Distel. It could lead to new ways to control the creature, which some consider the marine version of termites.

For LeChene, the shipworm study illustrates the power of the MIMS approach. Though it was originally designed to measure N15, MIMS can be used to detect a variety of stable and radioactive isotopes that play a role in a wide variety of physiological processes. LeChene is currently collaborating on more than a dozen studies using MIMS to examine various aspects of metabolism. In one project, he is working with HMS professor David Corey on a study of protein turnover in the inner ear.

According to Kuypers, “MIMS is truly an imaging breakthrough, whose application is only just beginning to yield information once considered inaccessible.”

—Misia Landau

top