Phage: The Phinal Phrontier

What phages are:

Our lab, the Phages Rock lab, works with bacteriophages (phages), and we work to isolate different types of phage as well as to analyze their genomes using computer programs. Phages infect bacteria in a similar way that viruses infect us, and once a phage is inside of a bacterial cell, it uses that bacterial cell to replicate itself. Depending on the phage’s genome, it can either integrate its DNA into the bacteria’s DNA, thus being replicated when the bacteria goes through DNA replication, or the phage can use the machinery within the bacteria to make clones before simply killing the bacteria. Some phages are able to infect many types of bacteria while some can only infect a few, and one type of bacteria can be infected by many different phages. This field of study isn’t new, but there is still much to learn, and there are thousands of phages yet to be discovered.

We work with phages that infect the bacteria Bacillus subtilis, which lives in the soil. In previous years, our lab has collected soil samples and extracted phage from them. In the past few weeks, we have been working with the phages that come from these soil samples. Much of our work deals with isolating different strains of phage, and our goal is to isolate new species of phage. Right now, we’re aiming to isolate 50-100 novel phages (but that number increases with every lab meeting). But, we don’t stop at isolating phages! We also do a lot of bioinformatics work to analyze different clusters of phage. Using several different computer programs such as DNAMaster, HHPred, and GeneMark, we look at phage DNA sequences to determine where genes are located in the DNA and what the functions of those genes are. But more on that later.

Wet Lab Work:

What we do at the beginning of our attempt to isolate phage is plate small portions of phage samples on a petri dish containing agar (a solidified substance for the bacteria and phage to interact in the petri dish) with small portions of various bacteria.  What we end up with are multiple petri dishes where the bacteria changes but the phage sample does not. After 18 to 24 hours of keeping these dishes in the incubator, we check to see if there are plaques, or clearings in the bacterial lawn, present on the different plates.  If there are plaques, then we know that the phage present in our sample infect that type of bacteria and we can attempt to isolate that phage on that strain of bacteria.  We will attempt isolation for each different morphology of plaque that is formed in a plaque search, assuming that different plaque formations will yield different phages.

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Figure 1. Plaque search from soil sample 280-A-7-8 by Marana Tso.

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Figure 2. Streak plate from soil sample 279-1-0-8 by Holly Wentworth.

 

Once we have identified the different morphologies of phages on our plaque search plates, we take isolated plaques, streak them out to ensure that each plaque represents one phage and not several phages, and then allow that specific morphology of phage to multiply. In the future, after we have built up our titers for the different phages, we will extract the DNA and send it off to be sequenced.  After the DNA has been sequenced, we will annotate the genome and call functions for specific genes through protein prediction software and by comparison to known phage genes. Isolated phages are also imaged using the transmission electron microscope.

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Figure 3. A transmission electron microscope of phage 015DV002.

Annotating:

Once we have a complete DNA sequence for a phage, we use computer tools to annotate the genome. We use a program called DNAMaster to determine where all of the genes in the genome are, and we look at information from other sources to determine which start codons actually start the genes. To determine the starts of the genes, we typically use information about what parts of the genome have the potential for protein coding, which sequence a ribosome is most likely to attach to, and which start similar phages use. Once we have determined where a gene starts and stops, we work to find the function. In many cases, genes have no known function. We know the functions of genes when someone else has done extensive lab work to determine the function. Even if there is no lab work, we can determine a putative function based on how close enough the gene is to others already determined. To determine the functions of genes, we often use programs such as BLASTP, HHPred, and Phamerator which compare the amino acid sequence of our gene to genes in the database to find a function based on similarity. We also use a program called Phyre2 which determines function based on the predicted  structure of the protein that the gene forms. Once the genomes have been annotated, we can begin performing comparative genomics.

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Figure 4. Screenshot of DNAMaster with the file for phage 015DV002 open.

Comparative Genomics:

The comparison and analysis of annotated genomes is known as comparative genomics. Phages are often compared to those that are similar in order to identify differences and similarities. This allows the phages to be classified as different phages or to be identified as a duplicate of another. An example of a possible difference between phages is in transfer RNAs (tRNAs). tRNAs are used to assemble proteins. Some phages code for tRNAs and between two similar phages there could be a difference in the number of tRNAs or the types of tRNAs that are called. The genomes can be compared in a multitude of ways, such as by sequence, gene order, gene function, and gene inclusion. Comparing the genomes of phages in different ways helps discern differences and similarities quicker than if the phages were compared simply between sequences or gene order. Computer programs like MEGA, which can align sequences and create phylogenetic trees, are often used in comparing genomes.

Comparative genomics is also useful when paired with wet lab work. For example, in evolutionary studies, a phage from the beginning of an evolutionary lineage and at the end of the lineage can be compared and differences can be identified. Experiments can also force phages to mutate and change the host range. That application is particularly pertinent if the ancestral phage has been fully annotated. Paired with comparative genomics, it is possible to compare phenotypic changes with genotypic changes. Similarly, if there are differences between the bacteria that can be infected by similar phages, comparing the genomes can shed some light on the differences in infection ability based upon the genes that may confer such capability. Furthermore, annotations and genomic analysis can be confirmed or denied through wet lab work.

*Title attributed to a tweet by Jason W. Shapiro.