PAPER Marine Environmental Genomics: New Secrets from a Mysterious Ocean AUTHORS ABSTRACT Karla B. Heidelberg Ocean microbes play critical roles in ecosystem dynamics and biogeochemical cycles. J. Craig Venter Institute For a number of reasons, these organisms have been hard to study; among other character- John F. Heidelberg istics, they are difficult or maybe impossible to culture. The recent application of cultivation- Institute for Genomic Research and independent genomic techniques to study bacterial communities has begun to fundamen- J. Craig Venter Institute tally change our views of microbial ecology and function. These approaches are providing more comprehensive insights into the structure and function of natural assemblages of microbial populations. Genomics-based technologies are revealing previously unknown groups of microorganisms and novel metabolic pathways, leading to a deeper appreciation oday we are witnessing an explosion in of the fundamental genetic and potential functional diversity of ocean microbes. When interest in marine microbial communities. evaluated in the context of observed ecosystem functions, we can begin to understand the TMicroscopic marine organisms, including complex interactions of individuals and populations with their physical and chemical envi- communities of phytoplankton, Bacteria, ronment. The continuing study and interpretation of the community genomic data will Archaea, protists and viruses, play critical roles require a close synergy among interdisciplinary researchers working throughout the world’s in the biosphere by participating in virtually ocean basins. Community genomic approaches have and will become a powerful tool for all of the Earth’s biogeochemical cycles and understanding marine microbial ecology in the future. thereby affecting geology, hydrology, and even possibly global climate change. These prolific and diverse microbial communities, while not powerful as these studies are, they do not pro- phylogentic genes. Amplification and se- seen by casual observers, by some estimates vide information on which microbes are re- quencing of the conserved regions of the 16S account for more than 90 percent of the ocean’s sponsible for specific biogeochemical processes. ribosomal RNA (rRNA) gene (part of the pro- biomass and 98 percent of the primary pro- Additional studies were designed to evalu- tein synthesis machinery found in all living duction in the marine environment (e.g. ate ocean function with the use of culturing cells), can provide the identity of the specific Whitman et al., 1998). Therefore, for hu- technologies. However, a significant challenge organisms in the sample. Initial application of mans to truly begin to understand the impact to our ability to study and understand these this methodology resulted in an explosion of of our activities on our environment, and to microorganisms is that the vast majority are information, providing for the first time a being to develop new tools to combat nega- not easily cultured on typical growth media, mechanism to evaluate microbial diversity for tive effects, it is important for us to under- the traditional approach in much of microbi- the > 99% of the microbial population that stand both diversity and function of the ma- ology dating back to the early days of medical could not be grown in the laboratory (e.g. rine microbial assemblages. microbiology and Koch’s principles. Cultur- DeLong et al., 1989; Giovannoni et al.; 1990; Traditional ‘black box’ microbial ecology ing methods were thought to be the necessary Pace, 1996, 1997). set the foundation for understanding ocean first step towards evaluating microbial diver- Despite the impact of these rRNA based function and taught us that unseen biology is sity and function, but we now know that surveys, a phylogenetic identification of a mi- of critical importance in the ocean. Through studying only microbial populations that can croorganism based solely on an rRNA sequence these studies we learned that microbes were at be grown in culture provides very little infor- does not allow inference of physiology, bio- the core of virtually all the biogeochemical mation on natural diversity or environmental chemistry, or ecological significance. Therefore, carbon (e.g. Carlson et al., 2001) and nutri- function (e.g. Beja et al., 2002a). Conse- the specific biological properties of abundant ent (e.g. Redfeld, 1958) cycles. For example, quently, over the past decade, scientists have uncultured microorganisms remain almost the global dissolved organic carbon pool is developed molecular tools to explore environ- entirely unknown. Another limitation of the estimated to be approximately 700 Pg C, a mental diversity without relying on culturing 16S rRNA sequencing technique is that it does value comparable to the mass of inorganic C technologies. The most commonly used cul- not distinguish between strains of the same in the atmosphere. Interaction of microbes ture-independent method relies on compari- species that may fill significantly different bio- within the dissolved organic pool could sons of homologous genes between organisms logical niches and habitats. For example, Es- strongly impact the balance between oceanic by using techniques such as polymerase chain cherichia coli, which persist in the human gut and atmospheric carbon dioxide. However, reaction (PCR) products typically targeting in a mutualistic relationship, may not be dis- 94 Marine Technology Society Journal tinguished from a closely related strain that erotrophic bacteria (Moran et al., 2004; trying to isolate a single organism, the total has acquired various toxins and is a deadly Giovannoni et al., 2005), and several community DNA is isolated en mass, the DNA human pathogen. cyanobacteria (e.g. Dufresne et al., 2003; is extracted, and this community of DNA Recent advances in genomic science have Palenik et al., 2003; Rocap et al., 2003)— serves as the template for sequencing. Once helped scientists to study cultured microor- are providing exciting information on the extracted, the DNA can be shotgun sequenced ganisms with much greater precision than detailed physiological and genetic controls as short insert libraries or analyzed with tar- ever before. Through the merging of DNA of photosynthesis, and the cycling of carbon geted large insert libraries (Figure 2). These are sequencing technologies and the use of newly and nitrogen in the world’s oceans. With re- powerful meta- or community genomics ap- developed computational methodologies a cent efforts funded by the Gordon and Betty proaches and allow for the gene compliment complete set of genetic blueprints of the or- Moore Foundation’s Marine Microbial se- of the entire community to be sequenced to ganism (the genomes) can now be ascertained quencing program (see Marine Microbial Ini- yield information on the potential biological in a very short period of time. Since the first tiative, http://www.moore.org/), the possible functions of the genes to be catalogued at a bacterial genome was sequenced a decade ago, addition of up to approximately 150 ge- very rapid pace. Analysis of small insert clones (Fleischmann et al., 1995), the number of nomes from cultured marine prokaryotes will from the Sargasso Sea (Venter et al., 2004), for fully sequenced deposited prokaryotic ge- greatly expand the existing marine genome example, yielded over one million previously nomes into public databases has grown tre- reference library. unknown genes, including almost 800 mendously (Figure 1). Genome sequencing However, perhaps the real potential of rhodopsins (a light absorbing molecule). In of microorganisms was initially prioritized to genomics may be in the newly developed addition to entire genome sequencing, studies study cultured pathogens, but recently there methods applied to study uncultured organ- using large insert libraries to evaluate detailed has been an expansion and focus on envi- isms—both prokaryotes and viruses. Several parts of organism’s genomes have helped make ronmentally important marine microbes. As key studies in 2004 (Tysen et al., Venter et al., important new discoveries, such as finding of expected, having the completed manually Tringe et al.) show that much could be learned a new form of phototrophy (Béjà et al., 2002b; curated genomes of key ocean organism ge- about entire communities of uncultured or- reviewed by Karl, 2002). It is clear that micro- nomes—e.g. a diatom, Thalassiosira ganisms by using high-throughput, shotgun bial communities have an extraordinary di- pseudonana (Armbrust et al., 2004), two het- DNA sequencing technologies. Rather than verse suite of mechanisms and pathways that are still yet to be discovered. Figure 1 As often is the case with new technologi- Publicly available completed genomes. Data reported by publication date, or if not published, the date cal advances, genomic technologies are being that genome data was deposited into the Comprehensive Microbial Database (CMR; http://cmr.tigr.org/ used by scientists based, for the most part, in tigr-scripts/CMR/CmrHomePage.cgi). Data for 2005 as of May. Data presented represent a total of 226 the developed world. It is tempting, for rea- genomes (199 Bacteria and 20 Archae and 3 Viruses). sons of convenience, to maintain research pro- grams in well-studied waters of one’s own coun- try. Ocean communities, however, function irrespective of political boundaries, and glo- bally, these new technologies have so much potential for informing marine management, sustainability,