Unpacking Baffling Bacterium

“For the longest time folks haven’t really paid that much attention to it except for it being this really quirky bacteria,” Moore says, noting that scientists were originally interested in Caulobacter for its unusual crescent shape. In order to help biologists better understand all of the surprising features of Caulobacter—for example, although it’s generally considered a non-pathogen, it’s caused infections in immunocompromised patients—Moore built a library of Caulobacter mutants for researchers to study.

Typically, when biologists want to study a bacterial process, they start randomly mutating the bacteria. Mutations can produce bacteria with weird appearances and processes; the hope is that one will have the change they’re looking for. “The issue with that is you need some kind of phenotype you’re looking for,” Moore says, “and you’re often looking for the most striking amount of change. You only learn about the ones that are successful and don’t learn about the rest of the population.” 

Instead of using the traditional (yet scattershot) process, Moore decided to make something targeted and useful for all the research groups interested in Caulobacter. The project turned into the first ordered transposon mutant library for the bacteria, called CauloKO. An article covering this is currently in revision at the Journal of Bacteriology.

To create the library, which was part of their doctoral work, Moore used transposons, or pieces of DNA with an enzyme that “basically hop around” and integrate into a genome. The long transposons act as a sort of knockout punch for a gene because if you stick this extra DNA into a gene, that gene fails. If you introduce transposons into enough cells, you can individually turn off nearly every gene in a genome. Combine that with a little DNA sequencing, and you have a collection of identifiable mutants—and Moore’s library.

The next step was identifying where the changes were happening in the mutants, which were located within 34,000 Caulobacter colonies in the wells of bacterial plates. A single plate of the bacterial library had 96 wells; each well in turn contained a different bacterial mutant. By placing 400 of those plates into a larger grid, you could identify each mutant by two things: its location within the larger grid of plates, and the smaller grid of wells within an individual plate.

It’s a technique called “knockout sudoku” with similarities to a sudoku board, where every row and column, as well as every square, has no repeat numbers. Moore arranged the bacterial plates containing the library of transposon knockout mutants into their own pools of materials to sequence the rows, columns, and “sudoku”-esque squares.

The proportions of the results identified where each sequenced mutant is located, in the same way you can identify where on your sudoku all instances of a certain number (say, the 4s) have to go. It’s a clever way to reduce the total sequencing for an entire genome from close to 30,000 to just 54.

The library covers about 70 to 80 percent of the total genome because many genes can’t tolerate transposon insertions. “Some of these things, if you knock it out, the organism just will not survive,” Moore says. “We call these essential genes.”

Two complete physical copies of the library exist, and Moore also created an online database so that every researcher who uses it can report errors; this way, the collection can continuously be improved and updated. “With buy-in from the community who do more screens in Caulobacter than I do now,” Moore says, “we can update the annotations and make the tool continuously useful in the future.”

Going forward, Moore’s lab at Oberlin—called The Gaybe Lab—is branching out to find and study other bacteria considered low-level pathogens. “We need to study these so if they become a problem in the clinic or in the world, then we already know a lot about them,” Moore says. In 2024, five students collected bacteria from places all over the Oberlin campus and are zeroing in on a unique specimen to study, working alongside three other students focused on public health analysis. This connection across typically disparate research disciplines reflects Moore’s interest in public health and the social determinants that impact infections—and their skill in creating community to impact change.