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Bacterial DNA uptake has IU scientists hooked
BLOOMINGTON, Ind.—Indiana University scientists have made the first direct observation of a key step in the process that bacteria use to rapidly evolve new traits, including antibiotic resistance.
Using methods invented at IU, researchers recorded the first images of bacterial appendages—over 10,000 times thinner than human hair—as they stretched out to catch DNA. These DNA fragments can then be incorporated into bacteria’s own genome through a process called DNA uptake, or horizontal gene transfer. The work was reported on June 11 in the journal Nature Microbiology.
“Horizontal gene transfer is an important way that antibiotic resistance moves between bacterial species, but the process has never been observed before, since the structures involved are so incredibly small,” said senior author Ankur Dalia, an assistant professor in the IU Bloomington College of Arts and Sciences’ Department of Biology. “It’s important to understand this process, since the more we understand about how bacteria share DNA, the better our chances are of thwarting it.”
The bacterium used in the study was Vibrio cholerae, the microbe that causes cholera. The bacterial structures used to catch DNA in the environment are extremely thin, hair-like appendages called pili.
“We chose to study Vibrio cholerae because it undergoes natural transformation readily in the environment. We also know a lot about the regulation of how Vibrio cholerae turns on the genes required for natural transformation, and so we can use that as a tool to make them take up DNA from the environment more often. It is also a very easy organism to work with and grows quickly, which makes it a great tool for studying basic biological questions,” according to Dalia and IU Ph.D. student Courtney Ellison.
Although scientists were aware that pili play a role in DNA uptake, direct evidence demonstrating how they work was lacking until this study. In order to observe pili in action, the scientists used a new method invented at IU to “paint” both the pili and DNA fragments with special glowing dyes. The team that developed this new method to label pili with dyes was led by IU Distinguished Professor Yves Brun and Ellison.
Dalia and Ellison explained that, “In order to record cells binding onto DNA using their pili, we first had to fluorescently label both the pili and DNA. While labeling DNA is very straightforward and can be easily achieved using commercially available dyes, we had to develop a method to label the pili because these tools did not exist. To label the pili, we genetically engineered them so that we could add a fluorescent dye that would bind to this modification. The kinds of dyes used to label the pili are called maleimide dyes. These dyes work by specifically binding to the thiol group of the amino acid cysteine, which we genetically engineered into the pili.
This method for labeling pili was developed by the first author of this study, Courtney Ellison in Yves Brun’s lab, and it was initially published last year in collaboration with Ankur Dalia in the journal Science.
The new study uses these dyes to reveal that pili act like microscopic “harpooners” that cast their line through pores in the cell's wall to “spear” a stray piece of DNA at the very tip. The pili then “reel” the DNA into the bacterial cell through the same pore. Dalia said the pore is so small that the DNA would need to fold in half to fit through the opening in the cell.
“It's like threading a needle,” said Ellison. “The size of the hole in the outer membrane is almost the exact width of a DNA helix bent in half, which is likely what is coming across. If there weren't a pilus to guide it, the chance the DNA would hit the pore at just the right angle to pass into the cell is basically zero.”
“[We] like to think of the pili as bacterial fishing rods,” Dalia and Ellison tell DDNews. “The pili are extended (or ‘cast’) and retracted (or ‘reeled’) back into the cell through tiny machines inside of the cell. The pili have specific proteins on their tips called minor pilins that they use as ‘fishing hooks’ to bind onto DNA, and when the pili are reeled back in they bring the attached DNA back with them.”
“One really exciting aspect of this research is that we found that the machinery used to retract or ‘reel’ in the pili is not actually required for this to occur. This was very surprising, since it was always thought before that this machinery was needed for retraction to happen,” they note. “This is another future direction that we are pursuing by trying to determine how the cells are able to reel in their pili when that machine is missing.”
According to Ellison and Dalia, “One area directly related to this work will be to try to understand how the protein associated with the tip of the fiber latches onto DNA and what interactions are occurring between those proteins and the DNA. We are very interested in the role and range of forces required for bending DNA to pull it into the cell, and we hope to learn more about these factors in the future by studying the machines that extend and retract the pili.
“Also, type IV pili can carry out very diverse processes, including attachment to surfaces, virulence and horizontal gene transfer. The pilus labeling method used here is allowing us to pursue many unanswered questions about these structures, so another major area of focus moving forward is to apply this method to study other pilus systems in Vibrio cholerae and beyond.”
“These are really versatile appendages,” Dalia said. “This method invented at IU is really opening up our basic understanding about a whole range of bacterial functions.”