A Tale of Phage Tails, a Range of Host Ranges, and Southerns from Out West

Bacteriophages (phages) are viruses that infect bacteria and are believed to constitute the most diverse group of organisms on Earth, with approximately 1031 phage particles (Hatfull 2008). To put this number in perspective, this many phage particles laid side-by-side would stretch roughly 10 million light years in distance (Suttle 2003).

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Transmission Electron Microscope image of a mycobacteriophage named Phamished, isolated by Sarah Brantley ’18 in the 2014 Biology 113 Introduction to Phage Biology course.

In our lab, we have been working on genomic characterization of novel phages by isolating, sequencing, and annotating their genomes to determine their gene functions. Of particular interest to us are tail fibers, which extend from the phage and are used for host adsorption and infection. Using phages we’ve isolated from Southwest National Park soil samples, we can further investigate phage genomics. By collecting at National Parks, we can ensure that our collection sites will be accessible in the future. For our research, we work primarily with phages of Bacillus subtilis, a gram-positive and spore-forming bacteria.

comparison

Using Southern hybridization techniques with probes developed by Katherine Boas’s ’16, we determine if we have phages in our samples that are similar genomically to phages we’ve already isolated. Immobilized target DNA is fixed to a nylon membrane and hybridized with a DIG-labeled probe and associated anti-DIG antibody coupled to an alkaline phosphatase – which attaches to the complementary DNA. By using this screening method we can identify and purify phages we haven’t seen yet and create a diversified Bacillus subtilis phage database.

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Visualization of Southern hybridization, where the probe binds to the complementary, immobilized target DNA, relaying a signal response (such as chemiluminescence with addition of an alkaline phosphatase substrate, CSPD) that allows for visualization of the binding.

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Applicability of Southern hybridization and probing procedure verified though probing of known phages: 010DV004, 015DV004, 031MP003, 056SW001, and SPP1.

Phages function as a fantastic model system for studying genomics, horizontal gene transfer, and mutation. Furthermore, this information can be used to study the ability of a virus to infect its host and expand its host range (the bacterial hosts which phages can successfully lyse). Performing bioinformatic analysis of these phages provides insights into evolutionary history and relationships among phages and other viruses, bacteria, and even eukaryotic organisms in some instances.

Of late, we (Madison Strine ’18, Alex Agesen ’18, Jenna DeCurzio ’18, and Rebecca King ’19) have been working with host-range mutants developed by Brianne Tomko ’16. These mutants were derived from 049ML001, a phage isolated on Bacillus subtilis T89-19 and originally collected from the Santa Catalina Mountains, Coronado National Forest, located north of Tucson, Arizona.

Phage 049ML001 belongs to a group of phages known as the SPP1-like phages. Three mutants were created using ultraviolet irradiation of the phage lysate, resulting in mutations within the genome that allowed for expansion of host range. Specifically, these mutants (M1, M2, and M3) can successfully lyse T89-03, a strain which 049ML001 infects with rather poor efficacy.

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We have finally grown enough phage (a few billion or so) and just performed DNA extraction for M1 and M3. We are still working on increasing the concentration of M2 to extract its DNA successfully. Sequencing of these mutant phage has the potential to identify genes of previously unknown function that are involved in determining host range. Specifically, if any of these mutants contain mutations within genes that possess an unknown function, we would then have data to support that these genes are involved in host infection and/or lysis.

As a side note, using these 049ML001 and our other SPP1-like phages from the Sonoran Desert, we have identified genes that were originally missed or misidentified in SPP1. Additionally, these phages have provided evidence that suggests some of the new genes that we observed in this group may have arisen either through splitting or merging existing genes.

We also recently attended the Evolution Meetings 2016 research conference in Austin, TX to present research that we have been working on since last summer.

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Top Row, Left to Right: Jenna DeCurzio ’18, Rebecca King ’19                                                                         Bottom Row, Left to Right: Dr. Veronique Delesalle, Natalie Tanke ’17, Madison Strine ’18, Alex Agesen ’18, Albert Vill ’16, Katherine Boas ’16, Brianne Tomko ’16, Dr. Greg Krukonis

At this conference, we presented on groups of phages we discovered that are closely related at the nucleotide level to each other and a phage previously known as a Singleton called SIOphi. These phages are also of considerable similarity to phiNIT1, Grass, and SPG24, other Bacillus phages. Here, a dotplot shows the degree of nucleotide identity across the genome among these phages, where a darker black line indicates higher nucleotide similarity.

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Using nucleotide similarity, we’ve grouped these phages into two groups: the SIOphi-like phages and the phiNIT1-like phages. The SIOphi-like phages are specialists, meaning they infect fewer than seven of the fourteen strains, whereas the phiNIT1-like phages are generalists, thus infecting a much wider range of Bacillus hosts.

Of particular interest to us was what causes this host range difference. After analyzing tail fibers, we found that 035J5004 (which infects more hosts than our other phages at 13 of 14 strains) has a tail fiber that has a different %GC content than its closest relatives [boxed in red in the genome maps below]. This same tail fiber also belongs to a different PHAM (a phage gene family assigned based on nucleotide identity) than its counterparts in its relatives. It’s possible that this gene is involved in the expanded host range we observed for this phage.

Furthermore, in the SIOphi-like phages, there was one tail fiber gene identified while in the phiNIT1-like phages there were two. Based on the nucleotide similarity of these genes, we postulated originally that two tail fiber genes in phiNIT1-like phages may have originated from the one SIOphi tail fiber gene – or vice versa. However, looking at %GC content we discovered that the story seems more complex because the average %GC of the two phiNIT1-like genes exceeded the %GC of the single SIOphi-like tail fiber gene. Ultimately, this suggests a different evolutionary origin for these genes despite their similarity at the nucleotide level.

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Also in Austin, Katherine presented on her aforementioned membrane probing and proof of principle in using Southern hybridization with known phages. Albert Vill ’16 presented on the comparative genetic analyses of six novel phages known as the 55kb phages, which although genetically similar to each other (and very different from any previouly sequenced phages) have distinct differences in their host ranges and evolutionary lineages. These phages also contain differences in major structural proteins, specifically in regions of tapemeasure (a protein which dictates tail length) and a sequence encoding the N-terminal domain of a major tail protein. Further investigation of these phages with mutagenesis may be carried out in the future to understand which genetic elements modulate the ability of a phage to infect bacteria.

Natalie Tanke ’17 presented on the reasoning behind the presence of tRNA genes in mycobacteriophage, phages that are isolated on Mycobacterium smegmatis. Numerous prior studies suggested that phages possessed tRNAs with rare codons that the host did not. However, Natalie’s findings did not coincide with this finding, since Trp (the most common tRNA in phages) is only coded for by one codon and, therefore, cannot be rare. As such, Natalie’s research suggests that amino acid bias rather than codon usage explains the presence of tRNAs. Furthermore, the location of tRNAs within the genome was found to be conserved with no significant impact on genome length. As a result, tRNAs must offer some benefit, since conservation of the tRNA location failed to show costs. As for how phages obtain there tRNA to begin with is still a mystery, since the native host of mycobacteriophages remains unknown. Her future research aims to look more at host range and tRNA possession among a variety of phages.

Soon, we will have full host-range data for some of our remaining SIOphi-like phages and all three 049ML001 mutants. We will also be performing electron microscopy to characterize the morphology of these phages in the next few days.

 

 

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