Diggin’ through the desert for a phage with no name…

With the stars still visible in the morning sky, we lifted our luggage into a van bound for BWI, our heads leaking the tailings of a Genetics final finished hours prior.  Our 7:30 flight to Las Vegas was packed with young adults and old couples, all chatting about what four-star hotels they were staying in and which casinos have the best slots.  We were Sin City bound, and we were likely the only people on the flight not heading straight for the strip.  In the eight days that followed, we, three students and our Chaco-clad professor, gallivanted across the American Southwest, sampling soil and salsas on the road from Vegas to Tucson.

 

Scientists of varied backgrounds have followed a similar trail for decades, observing and recording the unique geology and plant life of the region.  We admired the sun-baked landscapes and watched our footing among the less-than friendly flora, but our focus was drawn to the desert dirt, home to many members of the bacterial genus Bacillus.  The bacteria are merely the hosts for the primary reason of our trip, namely the tiny and plentiful viruses known as bacteriophages.

We are phage hunters.  And if the relevant literature gives any indication, our prey surrounds us.  Phage are in the water and soil, carried by the clouds and nestled in termite guts.  Where a prokaryote goes, you can bet its parasitic partner is not far behind.  With great abundance comes great variety, particularly when it comes to the genomes of these marvelous microfauna.  Our research as a lab deals dually with characterization of novel phages and processing of the existing stores of data.  While there are practical applications to our work, the whole field of viral biology is driven by pure scientific inquiry – how did this immense ecological variety arise? What genes account for the different infectious properties of these bacteriophage?  Though we’ve only scratched the surface, answering these questions will give insight to the evolutionary history of the Earth’s most plentiful organisms.

#dogreatwork

My name is Katherine Boas, I am a junior Biochemistry and Molecular Biology major and I work in the Krukonis/Delesalle lab, where we study bacteriophage ecology. Due to the miniscule size of bacteriophages just a spoonful of soil allows us to glimpse into the uncharted world of the phage. Bacteriophages are pivotal in the evolution of bacteria as well as the genetic changes that occur in soil, vegetation, and the oceans. Of these viruses that infect bacterial cells, we are specifically studying phages which infect Bacillus subtilis, a bacterium commonly found in soils where stress and starvation are common, such as the American southwest. Nine strains of Bacillus subtilis will be used to examine the host range of the bacteriophage in our soil samples. The DNA of four already sequenced Bacillus subtilis bacteriophages will then be used to probe the naturally occurring bacteriophage from the samples. What does it mean to probe the sample? Great question; a Roche DIG High Prime DNA labeling and Detection kit is used. Essentially, DIG (digoxigenin) is added to the genome of a phage through random priming and a probe is synthesized. DNA of phage to be probed is linked to a nylon membrane and then washed with various solutions one of which is the denatured probe. The DNA of the probe binds to complementary DNA linked to the membrane. Detection of the probe is performed using anti-DIG antibodies that only bind to DIG. The similarity of the phage DNA on the membrane to the DNA of the probe can then be visualized using CSPD (Chemiluminescent substrate for alkaline phosphatase). This technique is perfect for the assessment of phage diversity in the soil samples. By looking at the diversity of naturally occurring bacteriophage we can better understand how they interact with their hosts and thus understand the structure of these  communities. My focus this summer was to develop and refine the probing protocol.

Do ya DIG it?

Membrane Hybridization visualization

 

I am Albert Vill, a junior Biochemistry and Molecular Biology major working with Professors Krukonis and Delesalle.  While I have played a part in our lab’s Bacillus phage project, my personal work involves phages of Mycobacterium smegmatis (M. smeg).  So far, all characterized viruses of M. smeg contain double-stranded DNA (dsDNA) as their genetic material.  However, some bacterial hosts with known DNA phage have been found to have RNA-based viruses as well (e.g. DNA phage λ and RNA phage MS2 both infect E. coli).  We hypothesized that M. smeg has RNA phages, but that such viruses are less prolific than their dsDNA counterparts.  In an attempt to isolate an RNA virus from soil, I have modified an enrichment protocol to include hydroxyurea, a ribonucleotide reductase inhibitor used to prevent DNA replication without interfering with the synthesis of ribonucleic acid or proteins.  Essentially, the DNA phage present in the enrichment solution would not be able to reproduce, giving any RNA phage present a chance to proliferate.  Once a phage has been isolated and a high-concentration stock created – at least 10^8 viral particles per milliliter – I use electron microscopy as a screening process before continuing with additional tests.  A normal light microscope, sufficient for viewing animal and plant cells, is not powerful enough to resolve individual phage.  The average mycobacteriophage is only a few hundred nanometers from head to tail, meaning that 1500 phage could be arranged end to end around the circumference of a human hair.  Under the microscope, DNA phage have capsids (sphere-like structures containing the genetic material) with long, thin tails, whereas RNA phage consist only of capsids.  The preparation of the microscope samples is difficult, given that the tails can be easily “knocked off” of DNA phage, leading to false positives and necessitating procedural prudence and multiple samples.  If this isolation procedure is fruitful, further experiments will be done in coming semesters to extract RNA and reverse-transcribed DNA from the putative RNA-phage sample.

IMG_0006

DNA mycobacteriophage enveloped in phosphotungstic acid stain at 30000x magnification

 

I’m Brianne Tomko, also a junior Biochemistry and Molecular Biology major in the Krukonis/Delesalle lab.  This summer, in addition to collecting and working with the desert soil samples, I’ve been working with Bacillus phages isolated by Kendra Hayden ’12 in 2011 from soil collected in Tucson, AZ.  Kendra isolated 10 phages, whose genomes have all been sequenced and are in various stages of assembly.  In particular, I am working on annotating and characterizing one of these phages, K7.  Its relatively short (46,270bp), circular genome has been fully sequenced and assembled.  This is done by using the assembly program Newbler. It takes the tens of thousands of short sequences (reads) produced by the sequencing process and aligns them to put like sequences together and create longer contiguous sequences (contigs) that can be viewed in another program, Consed.

Newbler

Alignment of reads in Newbler

Sometimes the raw data will be assembled into one sequence, but other times, Consed will be used to put contigs together.  With Consed, you can look for similarities between the contigs to join them together into one sequence among other things.

Consed

Assembly View in Consed – the orange and black lines signify sequence similarity

Currently, I am annotating the genome to identify potential genes and their putative functions.  This is done by using various computer programs and databases to compare the genome in question to previously characterized and well-studied genomes.  K7 is closely related to another previously annotated Bacillus phage, SPP1, so I have also spent time studying SPP1 and comparing the two phages. These phages are also similar to another phage, PM1, which was sequenced in 2013.

Our next step is to grow more K7 to get mass spectrometry data.  The mass spectrometer separates proteins based on size and charge, and the output gives the mass and relative abundance of each protein in the sample.  This can also give information on the structure and function of the proteins. Phage genomes are very small, and from studying groups of similar phages, we know that the genes are tightly packed together, with very little unused space.  The structural genes in bacteriophages are pretty well studied and can be easy to identify, but the great majority of genes do not have easily detectable or well-studied functions, so there is a lot left to learn about these phages and their life cycles.

 

 

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One thought on “Diggin’ through the desert for a phage with no name…

  1. Pingback: The Phyllo Phages | Cross-Disciplinary Science Institute Summer Research

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