Experiments by the Garner Lab have yielded insights into how bacteria control their rate of growth of their external cell wall in response to the nutrients they are growing in. The cell wall is a mesh-like structure that surrounds bacteria and protects them from the environment. Researchers from the lab, led by research associate Yingjie Sun and MCO graduate student Sylvia Hürlimann, used high resolution microscopy and a novel analysis technique developed by Sun to examine how Bacillus subtilis bacteria control the number of machines that build the cell wall so that bacteria can grow. They discovered that a protein sensor plays an unexpected role in detecting levels of the building block needed for growing the cell wall, which then tunes up or down the abundance of proteins that build the cell wall. Their results appear in the latest issue of Nature Microbiology (PDF).
Bacterial growth is limited by nutrient availability, but the details of how bacteria control the rate at which they grow new cell wall in response to nutrient availability are largely unknown. In this study, the researchers focused on Bacillus subtilis and a compound called Lipid II, which serves as raw material for building the cell wall. They tracked activity of a group of proteins called the Rod complex, which inserts new strands of material into the cell wall.
“B. subtilis is a tiny bug,” Sun explains. “The diameter is only one micron, and there are so many rod complexes moving around the bacteria, it’s impossible to count them with normal diffraction limited microscopy…To get around this, we used high resolution imaging and an analysis technique I developed to count and track the number and speed of the Rod complexes in each cell. This allowed us to directly measure the cell’s overall cell wall synthesis activity and correlate this [activity] to cell growth. Ethan’s lab is a pioneer in the imaging of the Rod complex in bacteria.”
The researchers found that a protein called PrkC senses the Lipid II building block. When Lipid II levels increase, PrkC adds phosphate groups to a protein in the Rod complex, which increases the number active of Rod complexes, making the cell grow faster.
“What we found is that PrkC, which is a protein kinase, can sense the Lipid II precursor and then regulate the growth rate of the bacteria through the cell wall synthesis machinery,” says Sun. “That was a surprise, because previously, it was not reported that protein kinases, PrkC specifically, have any role in growth regulation.”
The Garner Lab team found that over-activating this PrkC-Rod complex pathway caused bacteria to grow far faster than wild type, even when they were growing in a minimal medium with few nutrients.
Reaching this conclusion required observing the growth of individual B. subtilis cells while measuring the activity of the very small Rod complexes within them. “By imaging one of the components of the Rod complex–the actin homolog MreB–and using an analysis technique that breaks the diffraction limit of light, we were able to characterize cell growth regulation at the single cell and single molecule level,” Sun says.
Sun says that understanding this pathway could help identify potential targets for antibiotics, which are largely aimed at destroying bacterial cell walls. Because PrkC is involved in many cellular processes, as well as cell growth, it could be a promising target as well.
This study gives the first insight into how bacteria couple the abundance of nutrients outside the cell to how fast they grow their cell wall, setting up new paradigms for the regulation of bacterial growth.
by Diana Crow and Ethan Garner
