A Genomic View of Oceanic Life

Last year, scientists discovered a unique, energy-generating, light-absorbing protein previously unknown to exist in oceanic life. They named the protein proteorhodopsin.1 The bacteria that harbor it are a distinct phylogenetic group known as SAR86. This year, scientists learned that as much as 10 percent of the ocean's surface is occupied by these proteorhodopsin-containing bacteria--as many as 1x105 cells per milliliter of sea water.2 The researchers, led by marine microbiologist Edward DeLong and his postdoc associate Oded Béjà at the Monterey Bay Aquarium Research Institute in Moss Landing, Calif., used what is sometimes referred to as "environmental genomics"--the application of bioinformatics and functional genomics to the study of ecology. Although scientists have been using this sort of large-scale bioinformatic approach to analyze genetic material of marine microbes since the early 1990s, Stephen Giovannoni, director of the molecular and cellular biology program at Oregon State University in Corvallis, says it wasn't until these proteorhodopsin papers were published that environmental biologists realized just how practical genomics can be for studying natural ecosystems. "It's the first clear evidence that this technology can be useful in terms of making discoveries about the way things work in an ecological system," Giovannoni comments. "It has really given that sort of approach a tremendous boost." Says DeLong, "Environmental genomics provides a much more realistic appreciation of the way the world really works." The proteorhodopsin discovery was a completely unpredicted outcome of a genomics approach to doing science, according to Giovannoni. Genomics, which he defines as "any effort to understand biological systems using large-scale analysis of DNA," resembles a fishing expedition. Researchers collect ocean water, bring it back to the lab, isolate large fragments of bacterial genomes, use a cloning vector to construct a DNA library, and then fish through the library for genome fragments with known phylogenetic characteristics. Once a fragment is identified, the genome of SAR86 for example, it can be isolated, sequenced, and examined for protein-encoding genes with known functions. And the analysis can occur without isolating even a single organism, a major boon for microbiologists because so many marine microbes are notoriously difficult to grow in the lab. Indeed, SAR86 has never been cultivated and is known only from genomic analysis. There are about 1 million microorganisms per milliliter of seawater, according to DeLong, but only about 0.1 to 1 percent of them can be cultivated. As a result, "99 percent of what's out there hasn't been biologically characterized," he says. Genomics "provides a window into a world we couldn't access before." The work of DeLong and Béjà, who worked in collaboration with John and Elena Spudich at the University of Texas Health Center in Houston, was well praised. "It's a spectacular thing to find proteorhodopsin in the ocean," says Giovannoni. Paul Falkowski, a biological oceanographer at the Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ, agrees. "It's a totally surprising mechanism for phototrophy in the ocean." (Phototrophy is the generation of metabolic energy from light.) Courtesy of Edward DeLong © MBARI A sample of oceanic bacteria as seen when using epifluorescence microscopy. The discovery of proteorhodopsin "has oceanographic significance," says Giovannoni. In some places, as much as 12 percent of surface water microbial life harbors proteorhodopsin, according to DeLong. And bacteria that do harbor them have an estimated 20,000 proteorhodopsin molecules per cell, which is consistent with what would be needed to make enough energy from light to live and reproduce. That these bacteria are so abundant and harbor so many proteorhodopsins suggests that proteorhodopsin-mediated phototrophic energy could be a significant source of energy uptake in the oceans. However, as Falkowski points out, the true distribution of proteorhodopsin-containing bacteria is unknown, especially in deeper waters, so it is difficult to accurately predict their role in the ocean's energy flux. Detecting Rhodopsin To detect rhodopsin, DeLong's lab uses bacterial artificial chromosome (BAC) cloning vectors,3 a technique he says is a spin-off from the Human Genome Project. BAC libraries are comprised of very large pieces of DNA, "as much as 10 percent of the entire genome of an organism," which can be rapidly screened for phylogenetic and other information. Not surprisingly, researchers find a lot of "house-keeping genes," says DeLong, which code for essential cellular functions. But in the case of SAR86, "there was one gene that really popped out. It was similar to bacteriorhodospin."1 Bacteriorhodopsin is a photon-driven ion pump embedded in the cell membrane bilayer.4 It's like a little bio-battery: when it absorbs light, its three-dimensional structure shifts shape and protons move through it across the membrane, creating an electrochemical membrane potential that generates ATP, the energy currency of life. Bacteriorhodopsin was discovered more than 30 years ago in the salt-marsh dwelling Halobacterium salinarum, an Archaean domain member. Since then, other rhodopsins have been revealed in the same taxa, and "up until two years ago, it was generally believed that this was the only place these microbial rhodopsin molecules were found," says John Spudich. To find a gene for rhodopsin in bacteria was surprising, adds DeLong. But locating a gene doesn't necessarily mean knowing how that gene is expressed. Indeed, says Falkowski, this is a genomics limitation; it's like identifying an insect but not knowing whether it's a mosquito, cockroach, or something else entirely. "We know what they are but not necessarily what they do," he explains. So the researchers took their discovery a couple of steps further. First, by expressing the gene in Escherichia coli, where "luckily it folded properly in the membrane," says DeLong, they were able to show that it did function as a light-driven photon pump.1 Then, Spudich's lab used laser-flash photolysis, a technique that monitors the protein's conformational changes as it absorbs light, and "showed that, indeed, this proteorhodopsin has a very rapid photocycle" characteristic of proton-pumping rhodopsins.1 Thus, they proved that it was more than just a gene; it was a protein. Despite its similar function to bacteriorhodopsin, proteorhodopsin was discovered in a new taxa, indeed a new domain (the Eubacteria) and is sufficiently different, Spudich says, to warrant the new name. Still, questions remained. They found the gene and expressed the protein, DeLong says, "but it was an observation in the lab. What happens out in the ocean?" So they headed to sea, collected more water, and, instead of analyzing isolated genome fragments, they cut to the chase and used laser-flash photolysis to analyze intact bacterial membrane preparations. They found direct biophysical evidence for the existence of proteorhodopsin pumps in marine microbial populations. "Everyone admires the fact that they were able to come around in full circle and effectively prove that what they had found in the lab is true in nature," Giovannoni says. Not only did they uncover proteorhodopsin everywhere they looked--in Monterey Bay, Antarctica, and Hawaii--they found extensive variation as well. In particular, the protein is spectrally tuned to different depths, absorbing light at different wavelengths depending on the environment. In Hawaii, for example, surface proteorhodopsins absorb light at about 530 nm, but the deep water (75 m below the surface) pigments exhibit a blue shift in their absorption spectra, absorbing light at about 480 nm. These absorption properties correspond to the spectral maxima at different depths, DeLong says. The blue-shifted proteorhodopsin variants are presumably better adapted to deeper waters where the blue light penetrates. "It's a nice adaptation to environmental light," Spudich comments. Environmental Genomics Unleashed For DeLong, the most exciting aspect of this research is that it shows how "we can use these genomic approaches to learn more about organisms in the world." They provide new tools that can identify characteristics of uncultivated marine microbes that "are evolutionarily very distinct from anything we've ever seen in the lab. There are major lineages out there in the ocean that we know nothing about." DeLong plans to continue using genomics in his search for proteorhodopsin-containing bacteria in the Pacific Ocean. Spudich's lab is working with Béjà, now on the faculty of the Technion Israel Institute of Technology in Haifa, Israel, to expand the search for proteorhodopsin in other parts of the world, including the Dead, Red, and Mediterranean seas. Spudich is also using bioinformatics to look for other, possibly novel members of the rhodopsin family in a diverse range of nonmarine taxa, including eukaryotes. Proteorhodopsin is only one of several rhodopsin proteins, all of which require light to function. And Spudich explains that he's unearthing rhodopsins in unexpected places, even in organisms as seemingly obscure as Cryptococcus neoformans, a toxic fungus that commonly infects HIV or other immunocompromised individuals. No one has examined the photophysiology of C. neoformans, he says, and it's not at all clear why it would harbor a rhodopsin. Spudich adds that, on the other hand, it's not surprising that rhodopsins are turning out to be so widespread, given that "light is the predominant force of nearly all environments on the earth." Even if proteorhodopsins can generate enough energy to sustain cellular life, cells require other sustenance, notes Spudich, like carbon and nitrogen, so they could still rely on other modes of energy uptake. Indeed, according to DeLong, "The next step is to figure out if these organisms are involved with carbon dioxide fixation." And once it's answered, it will "paint a more realistic picture of carbon cycling in marine microbial populations." Until the proteorhodopsin-containing SAR86 can be cultured or genomically analyzed to fish out nucleotide sequences for respiration or photosynthesis, other modes of energy uptake remain a mystery. Leslie Pray can be contacted at lpray@nasw.org References 1. O. Béjà et al., "Bacterial rhodopsin: evidence for a new type of phototrophy in the sea," Science, 289:1902-6, 2000. 2. O. Béjà et al., "Proteorhodopsin phototrophy in the ocean," Nature, 411: 786-9, June 14, 2001. 3. O. Béjà et al., "Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage," Environmental Microbiology, 2[5]:516-29, 2000. 4. L. Pray, "Sideline viewing of the BR membrane protein," The Scientist, 15[15]:22, July 23, 2001.

A Genomic View of Oceanic Life

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