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Brown lab awarded NSF grant to study cell growth in a pathogenic soil bacterium

Sept. 21, 2016

Dr. Pamela Brown

Dr. Pamela Brown is an Assistant Professor of Biological Sciences.

Pamela Brown, an assistant professor in the Division of Biological Sciences at the University of Missouri, received a three-year $624,000 grant from the National Science Foundation to study cell growth in the soil bacterium Agrobacterium tumefaciens.

“When we want to know how bacterial cells grow, we focus on how they expand their cell walls because that is the structure that allows them to maintain their cell shape and that is the structure that needs to elongate for a cell to get bigger,” Brown says.

The cell wall is required for most bacteria to survive, which is why it is a major target of a class of antibiotics used today. By understanding the molecular machinery that determines when and where new cell walls are built in this soil bacterium, the project has the long-term potential to identify new targets for antimicrobial drugs. It also has potential agricultural applications since the bacterium is the source of crown gall, an infectious disease common in rose, grape, stone fruits, shade and nut trees, among other plant species.

A major component of bacterial cell walls is a complex molecule called peptidoglycan. This molecule forms a tough but flexible lattice-like structure at the cell surface. To grow, the existing cell wall must be broken to allow insertion of new cell wall material. The molecular machinery responsible for coordinating where and when to break the existing wall and insert new material has been well studied in the model bacterium Escherichia coli. Brown says that A. tumefaciens and its close relatives, however, grow in a very different manner than E. coli, which is what makes it interesting.

“During elongation, E. coli inserts new wall material laterally at many foci along the side wall of the cell. In contrast, A. tumefaciens targets all of its growth to the pole of the cell. How and why it does that is really what we are trying to figure out,” Brown explains.

In this image, wildtype A. tumefaciens cells are labeled with fluorescent-d-amino acids (green) revealing the site of peptidoglycan biosynthesis at the cell poles during growth. Panels shown include differential interference contrast image of the cells (left), fluorescence image of the FDAA labeled cells (middle), and an overlay of the two images (right).

A. tumefaciens target all new cell growth (peptidoglycan biosynthesis in green) to the pole of the cell.

Brown explains that A. tumefaciens uses the same proteins to build new cell wall precursors but that most of the molecular machinery E. coli uses to direct this process to specific cellular locations is missing in A. tumefaciens. “What proteins comprise the cell wall biosynthesis machinery in agrobacterium? How is the biosynthetic complex properly placed at the pole during growth? What machinery keeps it there and prevents it from being active at other places in the cell? These are some of the key questions this project will attempt to answer.”

Unfortunately, the characteristic that makes the cell wall an attractive target for antibiotics also hampers research, says Brown. “The proteins required to build the cell wall and to regulate cell wall growth are essential for the bacterium to survive. You can’t just remove one of these proteins and ask what happens, because what happens is the cell dies.”

To overcome this challenge for this project, Brown will be using a genetic tool she adapted to study essential genes in A. tumefaciens. Rather than deleting the gene, Brown explains that this tool is sort of like a “dimmer switch” that allows her to slowly deplete a cell wall protein of interest in the bacterium. Then, using advanced imaging techniques and florescent tags, she can watch, in real time, what happens to the cell as that protein is depleted and before the cell dies. She reported on this so-called depletion strategy in the August 2016 issue of the scientific journal Applied and Environmental Microbiology.

“The ability to combine our ability to do live time-lapse imaging with our ability to remove target proteins of interest and watch what happens is a really powerful combination,” she says.

In nature, A. tumefaciens causes crown gall disease in flowering plants, including in many important crops. Infected plants develop tumor-like growths and suffer from yield reductions. The method by which the bacteria infect plants – transferring a particular segment of DNA from itself into plant cells – has been exploited by scientists to transform plants with DNA of interest. Brown says that increasing our knowledge about how this soil bacterium grows has potential practical applications in both of these realms.

“Certainly if we have a better understanding of the basic cell biology of A. tumefaciens, we may be able to devise new strategies to limit its negative effects on certain plants,” she says. “Likewise, we may be able to engineer better derivatives of A. tumefaciens to make it more effective as a biotechnology tool.”

In addition to being used for research purposes, the new grant will support the development of science curriculum to teach evolution using microbes as a model for lab exercises to high school and undergraduate students and future science teachers. Using a safe bacterium and inexpensive antibiotics, the curriculum will include a laboratory investigation that allows students to observe the phenomenon of antibiotic resistance first-hand by growing and plating bacteria.

An abstract of the project, titled “Mechanism of Asymmetric Growth of the Bacterial Plant Pathogen Agrobacterium tumefaciens,” is available on the NSF Web site.

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This movie of the growth and division of A. tumefaciens cells demonstrates the time-lapse microscopy the Brown lab will use as part of this new project.

Written by: Melody Kroll

Related research strengths:
Cell Biology, Molecular Biology, Plant Biology