Rohwer Lab

BioTools

Phage Proteomic Tree

Coral Microbes

Microbial landscapes on the surface of Porites compressa

SCUMS

Viruses and Phage

Viral Diversity
In almost all ecosystems that have been investigated, there are ~10 phage for every microbial cell (both Archaea and Bacteria), making them the most abundant biological entities on the planet. By killing microbes, phage greatly influence global biogeochemical cycles and because phage are species-specific predators they help maintain microbial diversity. Phage are also important mediators of genetic exchange, transducing ~5x1020 base pairs of DNA per day in the world´s oceans. Even though it is recognized that phage are extremely important ecosystem components, little is known about their diversity, adaptations, or evolution relationships.

Phage and movement of DNA between environment - A second way to assess uncultured phage diversity is to look for conserved genes by PCR or probing. Unfortunately, there is not a single locus in all phage that can be used in a manner analogous to the bacterial 16S rDNA. To get around this limitation, Rob Edwards and I developed a new phage taxonomy based on the available full genomic sequences. This taxonomy, called the Phage Proteomic Tree, placed phage into groups that closely match current views of phage biology and evolution (Rohwer & Edwards, 2002, J. Bact. 184, pp. 4529-4535). Using the Phage Proteomic Tree, we have identified conserved genes in the various phage clades that can serve as markers for these groups in the environment. We have used this approach to study the diversity of uncultured phage belonging to the T7-like Podophage clade. Not only have we found that this group is widespread in many environments, we have also found one particular sequence from this group of phage that is exactly the same in a number of environments (e.g., the same nucleotide sequence over ~650 bp!). This means that DNA can be transduced between vastly different environments by phage (Breitbart et al., 2004. Global distribution of nearly identical phage-encoded DNA sequences. FEMS Microbiol Letters. 236 (2) 245-252). We have now shown that phage from different biomes can propagate themselves on marine microbes (Sano et al., 2004. Movement of viruses between biomes. Applied and Environmental Microbiology. 70 (10). 5842-5846). We are now determining if other phage-encoded genes are also rapidly moving between environments.
Phage as environmental reservoirs of disease causing exotoxin genes - Many human diseases (e.g., anthrax, botulism, cholera, toxic shock syndrome, etc.) are caused by pathogens that produce exotoxins. Most of the genes that encode these exotoxins are encoded on phage or plasmids. These mobile elements can move the exotoxin genes among microbial hosts, converting avirulent microbes into pathogens. We have now shown that there is a pool of free phage in the environment that are a potential reservoir of phage-encoded exotoxins. Determining where exotoxin genes originate and under what conditions they infect avirulent hosts that cause human disease is crucial in our understanding and controlling the outbreaks of these diseases and detection of these pathogens. Currently we are expanding our survey to include hundreds of samples from all of the major biomes. Once we have determined where and when we can expect to find phage-encoded exotoxins, we will determine how these genes are being maintained in systems where the human diseases have not been observed (e.g., non-human reservoirs).

Roseophage SIO1 genome - In 2000, Farooq Azam (SIO), Anca Segall (SIO), and I published the genome of Roseophage SIO1, a marine phage. The genome showed that Roseophage SIO1 is evolutionarily related to coliphage T7. Other phage genomes that have been subsequently sequenced, by my group as well as others, have confirmed this finding, suggesting that there is a ubiquitous clade of marine phage related to coliphage T7 (see below). The manuscript describing this work was the feature article for the February 2000 issue of Limnology and Oceanography (Rohwer et al., 2000. The complete genomic sequence of the marine phage Roseophage SIO1 shares homology with non-marine phages. Limnology and Oceanography. 45 (2). 408-418).
Analysis of the Roseophage SIO1 suggested that this phage has a unique mechanism of initiating replication that is dependent on environmental phosphate concentrations. Since this finding we have shown that Roseophage SIO1 enters a pseudo-lysogenic state with its host under phosphate-replete conditions and then becomes lytic when phosphate concentrations decline. This finding has important ramifications for modeling phage-host dynamics in the marine environment where phosphate is often the limiting resource. Currently, my group is working with several members of the SDSU math department to predict how the lytic/lysogenic switch is controlled in the marine environment. This model is part of a greater research effort that includes Anca Segall (SDSU) and John Paul (U Southern Florida) that is melding genomics and environmental data to determine how the lysogeny/lytic decision is made in the ocean.

Uncultured viral diversity - One of the great unknowns in viral biology is what types of viruses there are in the environment. Most environmental viruses are phage and must be cultured on microbial hosts. Since it is estimated that 99% of the microbes in the environment cannot be grown under standard laboratory conditions, it is impossible to uses these traditional methods to study the majority of environmental phage. Recently, my lab has developed a method for building shotgun DNA libraries from uncultured phage. Using this metagenomic approach we have preformed the first analysis of uncultured viral communities from: 1) seawater (Breitbart et al., 2002, PNAS vol. 99, pp. 14250-14255), 2) fecal matter (Breitbart et al., 2003. J Bact. 85 (20). 6220-6223), and 3) sediments (Breitbart et al,. 2004. Proc Royal Soc B. 271. 565-574). Over 65% of the sequences in these libraries were not significantly similar to previously reported sequences, suggesting that most of the environmental phage belong to uncharacterized groups. Helped by mathematical models developed by collaborators in the SDSU math department, we found that the most abundant viral genome comprised 2-3% of the total population in both communities and that each community contained ~5,000-10,000 phage types. We are now applying this techniques to viral communities in extreme environments, human blood, and on corals. When completed this work will allow us to say what types of phage occur in all the major biomes of the planet and to compare the structure of phage communities in these environments. A practical application of this work is the identification of commercially interesting phage-encoded enzymes that we are finding by sequencing. This work is being done in conjunction with the biotechnology company Lucigen Corporation . This work is sponsored by NSF DEB 03-16518. Aquatic Phage Diversity. 08/03-07/07. $323,148. Rohwer (P.I.) and a grant from the Moore Foundation . The Global Virome. 11/04-10/07. $990,000. Rohwer (P.I.).