Each time the world girds itself for a new epidemic, scientists give the public a refresher course on how genes jump species, giving us flu strains that mingle DNA from birds, pigs and humans – and that pick up virulence genes along the way. Exactly how genes hop in and out of cells is still not well understood, but Briana Burton, a recently appointed Assistant Professor of Molecular and Cellular Biology at Harvard University, is elucidating some of the fundamental protein machinery underlying this process.
Basically, Burton studies the biochemistry of how cells solve the “oil and water” problem. The DNA double helix, the raw material of genes, likes the watery surrounding of the cytoplasm. The cell membrane, made of oily lipids, repels anything water loving, or hydrophilic. Yet bacteria are constantly passing huge pieces of hydrophilic DNA, often packaged as circular plasmids bearing new variants of genes to be shared with fellow microbes, through the hydrophobic membrane. Bacteria use DNA transport proteins, large complexes that function like machinery, to accomplish this still mysterious task.
Currently, Burton’s lab mainly focuses on one of these protein machines involved during one phase of spore formation in the Bacillus bacterium. But, just as she knows how to progress carefully but surely from toehold to toehold while rock climbing – her favorite recreation – she hopes the tools and methods she is developing to tackle this problem will help her reach the pinnacle of understanding how bacteria share resistance genes and how to interfere with this growing health threat.
Learning Science in Finnish
Burton began what may be a long scientific climb as a child living in the foothills of the Colorado Rocky Mountains but with roots in Finland, where she spent every summer. “Most of my summers were dedicated to learning physics from my grandfather,” Burton reminisced. “We would go on long walks through the Finnish forest everyday, and he would talk about his favorite subject, entropy.” Their discussions meandered from thermal physics through math and chemistry, inspiring her as an undergraduate at Northwestern University to take an intensive curriculum that spanned scientific disciplines in the Integrated Science Program.
Wishing to continue summering in Finland, she contacted Finnzymes, then a small biotech firm not far from her family’s home in Espoo. The director of R&D gave her a tour, offered her a paid internship, and that kindled her interest in biology, with a focus on basic science and goal of figuring out how things work. Upon graduating, Burton decided to devote her PhD work to protein machines. “I wanted to know the mechanisms of their interactions at the molecular level,” she recalled.
Shredding and Recycling
Burton took her Ph.D. at MIT, studying under Tania Baker and using E. coli to investigate a protein machine that should prevent the Phage Mu virus from being able to infect bacteria but instead allows it. To replicate inside the bacterium, the virus produces transposases. These transposases should naturally be targeted for destruction by a protein machine called the ClpX ATPase, which binds to doomed proteins and sends them to the “shredder” – protease proteins with “teeth” to chew up the doomed protein. (The large ATPase family of proteins uses the chemical energy from ATP and converts it to mechanical work, such as transporting other molecules.)
By studying these interactions, though, Burton discovered an alternate, unknown function of Clp ATPases. “Instead of always sending proteins to the garbage bin, they can also send proteins for recycling and reuse.” In the case of the transposases, that allows the virus to continue replicating and infecting other bacteria.
Transportation Across Barriers
As Burton prepared for a postdoc, she decided to focus on the oil and water problem of how protein machines transport DNA across cell membranes. “I was looking for labs that studied – at a biochemical, mechanistic level – how protein machines functioned, especially those that interact with nucleic acids like RNA and DNA. Since there were no labs studying the DNA transport systems the way I wanted to, I chose a lab that studied a related system.”
She joined Tom Rapoport’s lab at Harvard Medical School, which specializes in studying how other large molecules, polypeptides, are transported across membranes. Rapoport had developed an artificial membrane system and techniques for purifying the membrane-bound molecules and reconstituting this cross-membrane transfer in vitro for intensive molecular analysis. Burton wanted to replicate that system for DNA transport protein machines.
While working on that – which proves to be quite challenging – she also undertook in vivo work in bacteria to learn more about DNA transport proteins. “Bacteria have fewer checkpoints on cell division, so sometimes the membranes of the two daughter cells close up before the DNA is completely separated. Bacteria have DNA transport machines (also in the ATPase family) that ensure that DNA is not guillotined but instead is moved into the correct cell.”
She chose to study proteins from the non-pathogenic, spore-forming bacteria, Bacillus subtilis, even though no one in the Rapoport lab was working on it. “But there was an incredibly well established understanding of the Bacillus genome and cell biology and molecular function, thanks largely to the work of MCB’s Richard Losick,” Burton said. “But there wasn’t as much known about its biochemistry, so that was a good place for me to bring my background in mechanistic studies to the process.”
Fortuitously, David Rudner, a former Rich Losick postdoc, had recently set up his own lab at HMS, so he gave her the in vivo handle on Bacillus biology and how to do molecular genetics in the organism. DNA-Friendly Channels
One Bacillus DNA transporter protein, SpoIIIE, is particularly well suited for this study. SpoIIIE is essential when the cell divides asymmetrically and the smaller side forms a tiny spore that becomes environmentally stable even through extreme heat and desiccation. But since SpoIIIE is not needed for normal cell biology or regular cell division, Burton could study the effect of genetic changes on its function.
“Its job during sporulation is to move a huge chunk of the chromosome from the larger compartment into the smaller compartment so that each side receives a complete chromosome. We didn’t know whether the DNA is all moved before the membrane closes or after.”
She discovered that the membranes for each of the two new compartments have closed off before the DNA is completely ushered inside the spore, so that this large water-loving molecule must cross a double oily barrier. Her biochemical assays indicate that the SpoIIIE machine forms a channel, or pore, with a lipid exterior compatible with the membrane and a hydrophilic inner lining amenable to the nucleic acid. Finding out whether just one or more channels form during the process awaits refinements to the reconstitution of the in vitro system and new imaging equipment – and lab members.
Burton’s post-doctoral work uncovered enough clues about the architecture and mechanism of the DNA transporter’s complex that she was offered a position at MCB to continue that work in her own lab – which is adjacent to Richard Losick, her official mentor and Bacillus researcher extraordinaire. Also nearby is another new MCB member, Vlad Denic, and the Denic and Burton labs have joint group meetings. “Vlad is working on the a class of membrane proteins in yeast, so we use some shared techniques and approaches, just different organisms.”
Meanwhile, Burton is reaching for the next toehold of discovery – setting a reconstituted system to study how bacteria take up DNA from the environment. This fundamental research question holds some pretty large eventual clinical implications, such as explaining how antibiotic-resistant bacteria share their antibiotic resistance with each other.
“People are becoming more aware that there is a lot of exchange and communication among bacteria when they’re in multi-species communities,” Burton said. “And some of that genetic exchange is happening through these kinds of processes. We need to understand how the machineries work.”