he ocean harbors a mystery. Millions of tiny creatures invisible to the human eye thrive throughout the sea. Their function is unknown, because they cannot live outside of their native environment. And they are anonymous, because they have managed to elude capture by scientists.

The obscure organisms are marine bacteria. These little life forms are thought to make up 70 percent of the ocean’s living matter, or biomass. Microbes “occupy a peculiar place in the human view of life,” wrote Norman Pace, a biologist at the University of Colorado at Boulder, in Science magazine. Unless people are directly affected by microbes, as with disease, humans don’t pay attention to them. Yet all life on Earth greatly depends on the minuscule beings, which live in almost every known environment. Heat-loving bacteria flourish in boiling hot springs, E. coli bacteria live in animal intestines to help with food digestion, and radiation-resistant bacteria thrive in toxic dumps, converting hazardous materials into benign compounds.

Frustrated scientists know that marine bacteria exist, because they can see the organisms under a microscope. But when they try to grow the tiny life forms in the lab for study, the microbes die. So how can researchers catch and keep these elusive creatures long enough to find out what they are doing? This is a question that Ed DeLong, a biologist at the Monterey Bay Aquarium Research Institute (MBARI) in central California, has pondered for most of his scientific career.

DeLong uses a simple technique. He singles out one type of ocean microbe from a pool of water and decodes pieces of the bacteria’s genetic code. In this way, he is meticulously solving the puzzle of what specific micro-organisms are doing in the ocean, without growing the bugs in the lab. “I first got the idea from my old mentor Norm Pace, who said that you can start to identify organisms even if you can’t grow them,” he says. Now, after years of persistence, he has broken through a barrier. DeLong found that like plants, a large percentage of marine bacteria use sunlight to make energy. This discovery amazed the fields of oceanography and biology, because no one guessed that so many ocean microbes took advantage of the sun’s abundant gift of light.

These photosynthetic bacteria share a common trail with all cells. Ribosomal RNA (rRNA) genes are the code for pieces of a giant molecule called the ribosome. The ribosome is a protein-making factory found in all cells, from bacteria to humans. It works like a molecular assembly line, forging chains of proteins from the links in the cell. And each rRNA molecule is essential for its function. If the assembly line stopped, the cells would die, because proteins crucial to survival would not be made.

Little changes over millions of years have made rRNA genes a useful evolutionary marker. “All forms of life on earth have ribosomes, all ribosomes have the same ancestor, so you can compare ribosomal RNA genes,” DeLong says. Because of this conservation, researchers have used rRNA genes for decades to catalogue the Earth’s organisms on an evolutionary tree call the “tree of life.”

Recently developed technologies make decoding the entire genetic code – the genome – of an organism a snap for scientists. DeLong doesn’t require a live organism to get its genes’ sequences; all he needs is the DNA, or genetic material. Using a machine that unscrambles the genetic code, he finds the rRNA genes. Each organism’s rRNA genes vary slightly, but the overall sequence is retained. After comparing a new microbe’s rRNA genes with known ones, DeLong puts the organism on the tree of life.“There is a database of organisms that we can grow in the lab that are the backbone of the tree, a map,” he says. “rRNA genes are like a barcode, an identifier.”

Eight years ago, this technique helped him find that archaea, an ancient cousin of bacteria, are more abundant in the ocean waters than once thought. “DeLong’s discovery of the archaea in the ocean is a major contribution, it showed that there is this really divergent group that we didn’t know were so widespread,” says Jonathan Zehr, a microbial ecologist at the University of California Santa Cruz. Microbiologists originally thought archaea were restricted to extreme environments like thermal vents on the ocean floor. DeLong showed that the organisms make up more than 20 percent of the cells found in the ocean.

Merely investigating the abundance of organisms in the ocean wasn’t enough for DeLong. He wanted to know what the microbes were doing. Indeed, to imagine him resting on his laurels after a big discovery is like telling a puppy not to be curious.

So DeLong and his post-doctoral fellow Oded Beja embarked on a treasure hunt for clues in the mystery of ocean microbes. DeLong knew that he could figure out which bacteria he was looking at by their rRNA barcode, and he knew that genes are commonly arrayed along DNA strands in rows. So why not walk along the DNA strand and find out what other genes are next to the rRNA identifier? Maybe one of those new genes would lend insight into what some of the elusive bacteria were doing in the water.

DeLong and Beja’s first goal was to collect large quantities of marine bacteria, an easy task at MBARI. Perched at the edge of the ocean overlooking the Monterey Bay in the small town of Moss Landing, MBARI is a marine microbiologist’s heaven. Two large boats, called the Point Lobos and the Western Flyer, carry researchers out to the open ocean to collect samples on daily and weekly excursions. “The access to the ocean is unique at MBARI,” says DeLong. “In a couple of hours’ time, you are into very deep water.”

Ocean water samples contain millions of different kinds of bacteria. Beja and DeLong had to pass more than 1000 liters of water through a filtering apparatus to get a representative sample for study. “The filtration takes time,” Beja says, who has made many collections in Monterey, Hawaii, and Antarctica. The water pumps at a rate of two liters per minute, so it can take anywhere from two to ten hours to get enoughbugs for the studies. “It’s actually quite boring, and we eat a lot of ice cream while doing it,” he says.

Once the collection process was finished, Beja and DeLong took the samples back to MBARI. There, they gently removed the bacterial cell walls and opened up the organisms to extract the sticky, string-like genetic material. They used careful techniques to keep the strands long and intact. DNA is extremely fragile, and harsh treatment could easily result in it breaking apart like a wet piece of spaghetti pulled from both ends.

“When we first started we didn’t know what to look for,” Beja said. Indeed, the two researchers were flying blind, but in a short time they struck gold. From the DNA purified from the ocean samples they found a large piece containing the rRNA barcode, and walked along the rest of the strand looking for other genes. Each gene has distinct beginning and end points that are easy to spot. When they came to the first gene, they ran it through the database. Lo and behold, it was one that had never before been found in bacteria. And they found it in the first batch collected from the Monterey Bay. “We were lucky,” DeLong says.

The gene makes a protein called rhodopsin. When exposed to light, this molecule is activated. DeLong and Beja called their new discovery proteorhodopsin. It is the first light-sensitive molecule found associated with bacteria. Because proteorhodopsin needs sunlight to function, the finding is strong evidence that a large number of marine microbes use a form of photosynthesis to survive. This is a process previously only known to occur in plants and some archaea, but very rarely in bacteria. “Light for this (photosynthesis) is all over the ocean,” DeLong says.

The bacteria containing proteorhodopsin are called SAR86 on the tree of life. In prior work, DeLong showed that Sar86 are widespread throughout the surface waters of the ocean. The abundance of the bacteria suggests that light-sensing energy generation is happening throughout the ocean waters in a big way.

An important ingredient in the eyes of mammals, rhodopsin works in the only light-sensitive step in vision. When light shines on mammalian rhodopsin, it binds to a molecule called retinal to induce the vision process. Archaea also have a rhodopsin molecule, but with an added twist. Its protein both binds retinal and works to pump protons (the positively charged components of an atom’s nucleus) out of the cell to generate energy.

The discovery of proteorhodopsin was ground-breaking work. But DeLong remained unsatisfied, because he still could not grow the microbe that produced the new molecule. He was anxious to get the proteorhodopsin to work in E. coli, a bacteria easily grown in the lab. If it functioned in E. coli, the result would teach DeLong more about how the molecule worked. Of course, he still would not know if the light-sensing molecule was active in the wild. But the experiment would answer the question of whether proteorhodopsin turned on in response to light.

There was one potential problem. Lab strains of bacteria like E. coli often cannot survive the extreme surroundings where some marine microbes live. Like a fish pulled out of the sea and plopped onto land, taking a molecule out of a sensitive marine microbe and popping it into the mild conditions of the laboratory may kill it. If, however, the same fish is pulled out of the ocean and put it into a sale water tank, it will often live. It was possible that the new bacteria rhodopsin molecule would not work in the classic laboratory setting, and needed special treatment like the fish. If that were the case, it would bring DeLong back where he started, trying to cultivate bacteria that refused to live outside of the ocean.

Beja used molecular tricks to pop the rhodopsin gene into E. coli. Once the gene was in the bacteria, he added retinal to the bugs. Retinal is tiny enough for bacteria to suck through their membranes. Once the retinal was inside the bacteria, Beja shined light on the microbes. To his joy, the E. coli turned red, indicating that the rhodopsin molecule was binding to the retinal.

Beja then tested whether the rhodopsin was acting like a pump by measuring the number of protons pumped out of the cell. Amazingly, protons were pumped out, and he could measure the energy being generated. “This was surprising, because no one had ever done it, we made it pump!” DeLong says. Suddenly, DeLong had a major accomplishment on his plate. He and Beja had single handedly found a new function for ocean bacteria and had replicated that function in the laboratory.

Their findings were published in the September 15, 2000 issue of Science. The subsequent media frenzy surrounding the publication included news reports appearing in Discover, Scientific American, New Scientist and other magazines and newspapers.

Soon after he published the article, DeLong submitted a patent application for the bacteria rhodopsin gene. “It will be very handy for nanotechnology applications,” he says. The field of nanotechnology is based on using tiny molecules for research and medicine. The ability to manipulate the new bacteria rhodopsin in the laboratory makes it an ideal candidate for use in biotechnology. One possible application is to use the pumping apparatus to generate energy in E. coli. The bacteria would use the extra energy to make large amounts of proteins. Biotechnology companies often spend a lot of time and money making proteins when manufacturing drugs. The new mechanism could make the process much more efficient. “This opens many interesting doors that haven’t been opened very wide in microbiology,” DeLong says.

Along with interest from biotechnology came an offer of collaboration from The Institute for Genomic Research (TIGR) in Rockville, Maryland. The non-profit institute is lending its hand as a big genome facility to begin massive sequencing efforts on other ocean microbes that refuse to grow outside of the sea. “Ed’s recent paper is one of the greatest contributions to environmental biology,” says microbiologist Jonathan Eisen, one of DeLong’s collaborators at TIGR. Eisen is working with DeLong because he has also always been interested in the mystery of microbes that refuse to grow in the lab. “Three-fourths of the organisms on the evolutionary tree are bacteria, and if 99 percent are uncultured, then we know very little about the world,” he says.

TIGR researchers want to find new microbes to help deal with people’s increasing antibiotic resistance problems. Each year, billions of dollars are spent on experiments trying to generate new antibiotics. But the motherlode may be outside of the laboratory. Microbes have been fighting each other for millions of years. During this bacterial warfare, individual bacteria are creating antibiotics against its enemy microbes. Scientists hope to reach into this virtual medicine cabinet and identify new antibiotics using genomics. “It does not mean that the work being done in the lab isn’t very important, but this method just speeds up the process,” Eisen says.

The antibiotic dilemma is being tackled by scientist investigating another related mystery. Huge pools of un-named bacteria also live in the soil. So Eisen and TIGR are working with soil microbiologists to find new microbes in the Earth’s second greatest resource, land. “We are barely scratching the surface of knowing what makes up the biosphere,” says Robert Goodman, an environmental microbiologist at the University of Wisconsin at Madison. Goodman is on a quest for elusive soil microbes, with the intention of unearthing new antibiotics.

The “germ” of the idea formed in 1996, Goodman says. His fascination stems from early work by a Norwegian microbiologist named Torsvic who calculated and hypothesized the exact number of bacterial genomes on Earth. “My sense of that work, and soil microbiology, is that there were a lot of microbes about which nothing was known,” Goodman says.

Goodman has found, using techniques similar to DeLong’s, a number of molecules that kill microbes. The work is still in preliminary stages, but it looks promising. Goodman’s approach is slightly different from DeLong’s because he is looking for gene families rather than single genes. Most known antibiotic-producing microbes have antibiotic genes clustered next to each other on the DNA, like a string of little boxcars. When he finds a cluster of related genes, then he knows that he’s hit a pay dirt.

We are almost at the dawn of understanding microbiology in the global sense, Goodman says. DeLong’s work proved that genomics can pry open locked doors. But skeptics worry about relying too heavily on genomics, because a gene’s identity doesn’t reveal much about its function. For instance, a gene could be present in an organism, but never turned on in the environment. There are many examples of unused genes in humans, yeasts, and other animals.

Goodman says that the people who challenge the use of genomics are forgetting “that whether a forest is made up entirely of oaks or pine trees tells you a lot about the forests.” So if a gene is present, it may tell researchers something about evolution of the organism, or give clues to why the microbe lives in its environment. There will always be some who are unsatisfied until they can literally hold the organism in their hand, but the power of genomics is teaching scientists a great deal about microbes that refuse to be held.

For his part, DeLong doesn’t believe that his discovery is the answer to everything. “We’ve still got a lot to learn,” he says. Indeed, DeLong and Beja have now embarked upon a new journey. They want to get their rhodopsin to work in the ocean, to prove that the gene is turned on in the microbe’s natural environment.

“My real goal is to continue to learn about the ocean,” DeLong says. He remains committed to the question as to why many of the microorganisms in the ocean refuse to be grown in the laboratory. “Cultivation is one of the fundamental basics of microbiology,” he says. The age-old theories state that a scientist must be able to take an organism out of its natural environment, culture it in the lab, and then put it back in its original space to truly understand its function. It seems that DeLong will not rest until he can fulfill the first of those postulates with his mystery ocean microbes. He has been thinking of ways to do this, like growing the bacteria under blue light, a more natural environment for sea-faring creatures. But his current successes have also motivated him to continue with genomics.

Wherever his next voyage searching the ocean waters for mysterious microbes takes him, DeLong has definitely found his home at MBARI. Sitting in his chair facing a panoramic view of Monterey Bay, he smiles and says, “Yes, this is where I think large thoughts about little things.”