Like a mosquito on a summer evening, a bacteriophage is either feasting or in search of its next meal. But a bacteriophage isn’t interested in human blood and isn’t flying around your backyard. Bacteriophages — aka phages — are microscopic killers of bacteria. Wherever you find bacteria, which is nearly everywhere, you’ll find phages.
A phage is a virus, and the feasting begins when, like any virus, it attaches to its bacterium host and injects its DNA. The phage DNA hijacks the bacterium’s machinery and begins to reproduce itself. Soon, the bacterium is teeming with new phages that burst forth from the bacterium, destroying it. The hunt for a new victim begins.
Not long after Canadian scientist Felix d’Herelle gave a name to viruses that infect bacteria in 1917, he recognized their potential to treat disease. Using sewage, he isolated the dysentery phage and put it in solution. After he and other doctors in a Paris hospital drank a few pints to test it, they administered their phage solution to children dying from dysentery, who were cured the next day.
D’Herelle traveled across Europe and the Soviet Union with his microscopic miracles. When Alexander Fleming stumbled on penicillin in 1928, however, the world had a magic bullet for bacterial infections. Except in a few countries, the use of phages declined into oblivion.
Today, with fast evolving, antibiotic-resistant bacteria, phages are back in the spotlight. With bioinformatics tools, researchers are seeking to understand them at the level of DNA and genes.
In recent work with Pittsburgh Supercomputing Center computational resources, and an important boost from PSC training, Aleisha Dobbins of Howard University and coworkers at the Pittsburgh Bacteriophage Institute, reported the complete genome analysis of a well-known but little understood phage. Her results — reported in the Journal of Bacteriology (April 2004) — reveal the entire DNA sequence and identify the genes of the SP6 bacteriophage.
With phages, the sheer numbers are almost scary — 10 million populate a milliliter of seawater, about 50 drops. Phages comprise the majority of organisms on the planet, and through the recycling of carbon in the oceans may be responsible for up to a quarter of the planet’s energy turnover.
Electron micrograph of two SP6 virus particles, the roughly hexagonal-shaped head is about 50 nanometers in diameter.
“When people hear the word ‘virus,’ they think trouble,” says Dobbins. “But phages kill bacteria and have no effect on humans. They can be used in addition to antibiotics. With interest in phage therapy resurfacing, it’s important to do sequence analysis and map the genes of more phages.”
Research in phages is also part of the effort to defeat human viral disease. Having all the genes they need to replicate themselves, phages are similar to viruses that invade humans, but easier to study because their hosts are bacteria, not humans. If researchers learn how phages assemble their protein houses, called capsids, they may develop the means to dismantle them. Without the capsid shells, both phages and human viruses are harmless bits of floating DNA.
The SP6 phage in particular attracts attention because of its host. Phages are picky parasites. Each one invades a particular bacterium, and SP6 goes after Salmonella, the nasty bacteria that dwell in raw meat and cause food poisoning. While SP6 hasn’t been yet been used to treat food poisoning, it is widely used in biotechnology.
SP6’s RNA polymerase, an enzyme that transcribes DNA into RNA, is commonly used in genetic technology to modify and clone the DNA sequences of bacteria. Despite wide use, SP6 had not been sequenced and most of its genes had not been identified before Dobbins’ work.
As a Ph.D student working on her dissertation, Dobbins planned to focus on one of SP6’s genes. Her plans became more ambitious, however, when she went to a PSC bioinformatics workshop, led by PSC scientist and sequence-analysis expert Hugh Nicholas. Through the workshop, Dobbins gained the ability to tackle the much larger project of the entire SP6 genome.
“Through this workshop,” says Dobbins, “I gained knowledge of the bioinformatics tools I needed to sequence the genome. And I learned how to use software to identify the termination sequences.”
From July 12 to 23, 2004, PSC hosted 19 faculty and staff from nine universities for its two-week course, “Developing Bioinformatics Programs.” PSC scientists Hugh Nicholas (1st row center) and David Deerfield (2nd row right) led the course. Five interns from three universities stayed at PSC for five weeks to continue work on their research projects.
The tools of bioinformatics, which marry information science and statistics with the life sciences, allow researchers to understand biological systems like never before. But researchers need to learn about these new and rapidly improving tools. PSC’s “Developing Bioinformatics Programs” course introduces faculty and graduate students from minority-serving institutions to the computational, mathematical, and biological issues of bioinformatics.
“Bioinformatics computer programs in general involve implementing a mathematical model and comparing the model with the data to see how they relate to each other,” says PSC scientist Hugh Nicholas. “The most common bioinformatics task involves taking a sequence from a biologist’s laboratory and comparing it to all sequences in the database looking for relationships according to a mathematical model of sequence evolution.”
The two-week workshop trains faculty who plan to establish an introductory bioinformatics course at their home institution, and graduate students, such as Aleisha Dobbins, to use bioinformatics tools to complete a research project. The course is sponsored by a grant from the Minority Access to Research Careers Branch of the Branch of Division of Minority Opportunity in Research of the National Institute of General Medical Sciences. It grew out of a bioinformatics workshop originally developed through support from NIH’s National Center for Research Resources, which also supports Tourney, PSC’s sequence-analysis computer, used during the workshop and by university students in courses developed through the workshop. Tourney is available to support bioinformatics course work at any U.S. academic institution.
Nicholas introduced Dobbins to researchers at the Pittsburgh Bacteriophage Institute (PBI) at the University of Pittsburgh. Dobbins and PBI co-directors Roger Hendrix and Graham Hatfull decided that rather than examining one gene, it made sense to sequence and examine the entire genome. This would allow them to compare SP6 with other well-known phages and, perhaps, to draw conclusions about SP6’s evolution, information that relates directly to the ability of bacteria to evolve and defeat antibiotics.
Phages and bacteria evolve in conjunction, bound together in the race to outwit one another and survive. Through this evolutionary drama, phages introduce new genes into the bacteria population. “Most human pathogens are as toxic as they are because of genes that were brought in by phages,” says PBI’s Hendrix. “There’s a lot of interest in what this population looks like and by comparing them to each other we can start to see how the population evolved up to where it is now.”
At PBI, Dobbins sequenced SP6’s entire genome of over 40,000 nucleotides, the building blocks of DNA. She also identified some of the genes and their order. With training from Nicholas and the PSC workshop, she used Tourney, PSC’s sequence-analysis computer, to identify the terminator sequences — regions of the genome that signal RNA polymerases to stop transcribing and disconnect. With Tourney, Dobbins also compared SP6 sequences with databases of known phage gene sequences and thereby identified SP6’s genes.
Dobbins identified SP6 as being part of the T7 phage family, which includes T7 and T3, two of the most well researched phages — a family relation that had been suspected, but not verified. Because of their similarities, Dobbins used a template of the T7 RNA polymerase, which is also used in genetic technology, to build a model of SP6’s polymerase, the gene she hoped to examine in her original research plan.
Phages in the T7 family presumably evolved from the same ancestor as SP6, and have many similarities in sequence and gene placement. But there are many family mysteries. Through comparative analysis, Dobbins found that one sequence of genes appearing in the same place in most phages in the T7 family was in a much different place in SP6. The group is not only in a different place, but reversed in order. As with any research, answers spark new questions.
Dobbins’ work will feed an ongoing discussion about phage evolution. While some believe that they evolved from a common ancestor millions of years ago, others argue that similar structures arose independently, or diverged more recently.
“The evolution of bacteriophages has not totally been traced,” said Dobbins. “We don’t know how they have evolved or what kind of effect this phage had on bacteria. We completed the sequence and found that it had 52 genes, and of the 52, 64 percent are unique to SP6. A lot of additional work needs to be done to identify the function of those genes.”