Mission Possible: The Mystery of tRNA Genes in Mycobacteriophages

     Mission Report


Agents:

Natalie Tanke

  • Rising sophomore, biology major
  • Future career goals: continue research into grad school
  • Current Location: Krukonis and Delesalle Lab

Celina Harris

  • Rising sophomore, chemistry major
  • Future career goals: continue research into grad school
  • Current Location: Krukonis and Delesalle Lab

Operation:

Collect further information on bacteriophages. See future reports from agents Vill, Boas, and Tomko.

Background:

Villain ‘SUPER-BUG’ has been popping up in news reports once again. More bacteria are developing antibiotic resistance. The public is in a frenzy. Attempts to pull bacteriophages, viruses that can lyse bacteria, to our side have been futile.

What is a bacteriophage?

A phage is a small virus that infects bacteria and relies on the bacteria to replicate. Phages are considered the most diverse entities on Earth and lack of knowledge about them has many researchers curious – this is where we come in, we work to acquire more knowledge on these potential allies. Phages can enter one of two life cycles. Phages in the lytic life cycle lyse and kill the bacterial host whereas phages that enter the lysogenic cycle integrate into the hosts – where they are now called prophages. The phage relies on the host’s machinery for survival and replication (Figure 1).

Figure 1. The lytic and lysogenic life cycle stages of phage replication (Reece et al., 2011, pg. 386).

Figure 1. The lytic and lysogenic life cycle stages of phage replication (Reece et al., 2011, pg. 386).

Since phages rely on the host’s machinery, including its tRNA genes, we are curious as to why some phages possess tRNA genes (Figure 2).

 Figure 2. tRNA genes are responsible for transferring the appropriate amino acid to the growing polypeptide (protein) chain. Translation of the mRNA into a protein occurs at ribosomes.  The matching of the anticodon, labeled above, to the codon of the mRNA allows the correct amino acid to be added to the growing chain (Reece et al., 2011, pg. 338).


Figure 2. tRNA genes are responsible for transferring the appropriate amino acid to the growing polypeptide (protein) chain. Translation of the mRNA into a protein occurs at ribosomes. The matching of the anticodon, labeled above, to the codon of the mRNA allows the correct amino acid to be added to the growing chain (Reece et al., 2011, pg. 338).

Other agents:

This mission comes from the work agents have been doing through the SEA-PHAGES program of the Howard Hughes Medical Institute (HHMI). The SEA-PHAGES program is offered to students across the world at over 70 campuses. The program involves wet lab work in which students obtain a soil sample, typically a spoonful of soil from somewhere on their campus, enrich the sample, and then attempt to isolate a novel bacteriophage. At Gettysburg College, as well as most campuses, the phage is plated/isolated on Mycobacterium smegmatis mc2155. However as the program grows more schools are exploring other bacterial hosts.

Additionally, there is also a bioinformatics side to this program, which is very intensive as well. One phage isolated by the class is sequenced. Then students annotate the phage’s genome to determine where genes start and stop, the length of genes, and potential functions. Students are also given the opportunity to explore other online bioinformatics tools. This is an excellent way to integrate concepts from lecture and provide thought-provoking discussions.

Each school runs the SEA-PHAGES course slightly differently. While some schools may integrate lab and lecture, others are strictly lab based. At Gettysburg College, the goal is to make the course as beneficial and fulfilling to first-year students as possible. Visit phagesdb.org to check out all of the currently sequenced and annotated phages that have been isolated on Mycobacterium smegmatis mc2155. Over 650 mycobacteriophages have been sequenced, annotated, and divided into numerous clusters.

Our mission

Through the bioinformatics section of the program, our current mission was revealed. Our novel phage, Tiffany, a member of cluster A, contains three tRNA genes. Many members of Cluster A contain tRNA genes. Phage tRNA genes code for only a few of the 20 amino acids. While Mycobacterium smegmatis mc2155 contains 47 tRNA genes, we observe considerably fewer tRNA genes in cluster A phages, ranging from 1-5 genes within the phage genome (Figure 3).

Figure 3. Cluster A phages are divided into 11 subclusters based on how similar their nucleotide sequences are. Within each subcluster some phages contain tRNA genes, responsible for transporting the correct amino acid to the protein being built. The above chart indicates the proportion of phages in each subcluster that contain particular tRNA genes. Subcluster A4 is the only subcluster that has no phages with tRNA genes. Tryptophan is the amino acid most commonly coded for by cluster A tRNA genes.

Figure 3. Cluster A phages are divided into 11 subclusters based on how similar their nucleotide sequences are. Within each subcluster some phages contain tRNA genes, responsible for transporting the correct amino acid to the protein being built. The above chart indicates the proportion of phages in each subcluster that contain particular tRNA genes. Subcluster A4 is the only subcluster that has no phages with tRNA genes. Tryptophan is the amino acid most commonly coded for by cluster A tRNA genes.

Our Goal

  1. Determine why phages contain tRNA genes
  2. Propose several phylogenetic hypotheses for tRNA gene possession in subclusters A2 and A3

Our Report Back to HQ:

            We attended a research symposium in Ashburn, VA at HHMI’s Janelia Research Campus in June. This was a three-day exposure to new and upcoming phage research. It also gave us the opportunity to share our research and brainstorm ideas with other universities and colleges participating in the SEA-PHAGES program. During the symposium we attended several lectures, given by both students and faculty, shared our class poster, and gave a talk to an audience of 250 people. The symposium required extensive preparation and additional research that extended beyond our work during the 2013-2014 school year. This symposium was an incredible and eye-opening experience. It was wonderful to be around so many people who shared our passion for mycobacteriophages and it was an inspiring opportunity to look further into cluster A phages.

Goal 1

To look into why phages contain tRNA genes, we focused on codon usage. For instance, our novel phage Tiffany contains the tRNA gene for the amino acid asparagine. Asparagine is coded for by the codons (sets of three nucleotides) AAT or AAC. Since this tRNA gene attaches to the codon AAT, we would expect that the AAT codon would be used significantly more than the AAC codon in the genomes of phage with this gene. To test this hypothesis, we exported the codon usage for several phages using a program called DNA master. This allowed us to look at every gene within the genome and how many times each codon was used by each gene. We then performed several t-tests and contingency chi-square tests to see if there was a significant difference in codon usage in phages with and without tRNA genes, in phages with varying numbers of tRNA genes, and in structural vs. non-structural genes. The only instance in which we found a significant difference in codon usage was in structural vs. non-structural genes within phages of subcluster A3. The codon for asparagine was used significantly more in the non-structural genes of phages with tRNA genes than in those without tRNA genes. We are still interested in why this is and will have to perform future tests.

Goal 2

We are also interested in how phages come to have tRNA genes in their genomes. We focused on subclusters A2 and A3 since they have large numbers of members as well as both phages with and without tRNA genes. In order to generate phylogenetic trees, we ran the tapemeasure gene, the longest gene within the phage genome, through a program called MEGA. The amino acid sequences were aligned and then trees were generated based on the best-fit model. Subcluster A3 provides a simple story of tRNA gene possession. There are 37 phages in subcluster A3, 36 of which contain 1 to 3 tRNA genes (Figure 4). For subcluster A2, several different phylogenetic hypotheses can be proposed. Subcluster A2 contains 29 phages, 25 of which contain 1 to 5 tRNA genes (Figure 5). Phages may possess tRNA genes due to ancestral conditions or horizontal gene transfer. We are working on blasting genes against other phages to look for similarities or dissimilarities with tRNA genes. Note: these trees are based on data on phagesdb.org as of May 2014. Since then several additions as well as changes have been made to the database.

Figure 4. Phylogenetic tree for subcluster A3, based on the tapemeasure gene. The colored circles to the right of each phage indicate which tRNA genes each phage contains. The numbers listed on the branches are the probabilities for how likely it is that those branches exist. The A3 subcluster divides nicely into 4 clades. It is probable that the ancestral condition was a phage with three tRNA genes -for asparagine, leucine, and tryptophan - and that each of these tRNA genes was lost in turn (as indicated by X on tree) in some subset of this cluster. This scenario suggests only three losses of tRNA genes in this subcluster.

Figure 4. Phylogenetic tree for subcluster A3, based on the tapemeasure gene. The colored circles to the right of each phage indicate which tRNA genes each phage contains. The numbers listed on the branches are the probabilities for how likely it is that those branches exist. The A3 subcluster divides nicely into 4 clades. It is probable that the ancestral condition was a phage with three tRNA genes -for asparagine, leucine, and tryptophan – and that each of these tRNA genes was lost in turn (as indicated by X on tree) in some subset of this cluster. This scenario suggests only three losses of tRNA genes in this subcluster.

Figure 5. Phylogenetic tree for the A2 subcluster. Several ancestral conditions are possible based on the distribution of tRNA genes. This figure depicts gains and losses based on the ancestral condition of no tRNA genes. Other ancestral conditions are possible: the ancestral phage may have contained glutamine and tryptophan originally, the two most frequently seen tRNA genes, or may have contained the same 5 tRNA genes as D29, a unique A2 phage since it contains 5 tRNA genes. All scenarios suggest multiple gains and losses of tRNA genes in this subcluster.

Figure 5. Phylogenetic tree for the A2 subcluster. Several ancestral conditions are possible based on the distribution of tRNA genes. This figure depicts gains and losses based on the ancestral condition of no tRNA genes. Other ancestral conditions are possible: the ancestral phage may have contained glutamine and tryptophan originally, the two most frequently seen tRNA genes, or may have contained the same 5 tRNA genes as D29, a unique A2 phage since it contains 5 tRNA genes. All scenarios suggest multiple gains and losses of tRNA genes in this subcluster.

 

Summary:

            We report that Janelia was a success! We are currently extending our research across all cluster A phages.  There are so many interesting trends and observations we can make in regards to tRNA gene distribution. The list is plentiful and there will always be new and exciting avenues to explore!

 

…but there will always be more research to do!

 

Reference:

Reece, J., Urry, L., Cain, M., Wasserman, S., Minorsky, P., & Jackson, R.

(2011). Campbell Biology (9th ed.).  San Franciso, United States of America:

Pearson Benjamin Cummings.

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One thought on “Mission Possible: The Mystery of tRNA Genes in Mycobacteriophages

  1. Pingback: Diggin’ through the desert for a phage with no name… | Cross-Disciplinary Science Institute Summer Research

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