To suss out which stretches of the Caulobacter crescentus genome are necessary for its function, Lucy Shapiro and her group at Stanford University oversaturated it with transposons. If the transposon lands in, and disrupts, a region of the Caulobacter crescentus genome that is essential for bacterial function, then that clone cannot be grown on rich media, while bacteria with non-essential disruptions can. "We are looking for the dog that doesn't bark," Shapiro says.
In all, her team identified 1,012 essential features, including 402 regulatory sequences and 130 non-coding elements, at a resolution of 8 base pairs.
To do the disrupting, Shapiro's PhD student, Beat Christen, engineered a Tn5 transposon to carry an outward-pointing inducible promoter at one end. "You want to use the transposon which can target any of these DNA bases in the entire genome and can probe information at every DNA letter," Christen says. "The beauty of this Tn5 element is that it has almost no site specificity, which allows you to get to almost every single base pair in the bacterial genome." Then he isolated DNA from each insertion site, amplified it by PCR, and sequenced it using Illumina paired-end sequencing to determine the location and orientation of the insertions. From that, he identified 480 essential open-reading frames.
This approach, Shapiro says, allows for high-throughput characterization of genome function, which, she adds has been the "Holy Grail."
"We have now identified every promoter regulatory region, every regulatory binding site, every small RNA, every answer, everything, every part of every gene that is essential," Shapiro says, adding, "we can look at the entire coding sequence for a protein and know down there at the C-terminal end of the encoded protein how much is essential." Indeed, the researchers found a number of protein-coding genes that didn't need portions of their C-termini to be viable.
In addition, the researchers identified 90 essential small genome elements whose function is unknown. "We don't know what the hell they are," Shapiro says. "They make no sense in the context of anything we know about current genome annotation." She plans to follow up on those small elements to figure out what they do.
Shapiro adds that this strategy to identify the essential genome may be applied to other bacterial genomes, as long as they have been sequenced. For instance, she notes that the methodology could be used to find the essential genome of pathogens like Salmonella or Agrobacterium.
Further, knowing the essential genomes of bacteria can help synthetic biologists in their work. "You have to understand every single regulatory element, every feedback loop, every circuit element that allows you to turn a gene on or off in sequence, in place, in time, and once you have what we have now done for Caulobacter in any system, it is not just a step forward. It's a leap forward for synthetic biology," Shapiro says.