Researchers have discovered how acids on the surface of bacteria give these microscopic organisms their characteristic "rod" shape—by keeping an enzyme at bay that would otherwise turn the cylindrical cells into shape-shifting blobs.
The findings, published in Nature Microbiology , provide a new understanding of how bacteria control their growth and offer insights into the nature of Earth's earliest life forms. The study also points to a strategy for overcoming antibiotic resistance by targeting wall teichoic acids, the enigmatic molecules that coat the surface of certain bacteria.
Bacteria occur in a range of shapes, with rod-shaped bacteria being by far the most common. The shape of bacteria is important medically since the cell wall—bacteria's rigid exoskeleton—determines cell shape and is the target of frontline antibiotics.
"A bacterium's shape dictates how it grows, how it divides, and how it interacts with its environment," said Felix Barber, who led this research as a postdoc in NYU's Department of Biology and is now an assistant professor at The Ohio State University. "Understanding the factors that give rise to the shape of bacteria are important because those same factors are also what we want to inhibit with antibiotics."
Bacillus subtilis (B. subtilis) is a rod-shaped bacterium that is naturally found in soil and in the gut. Considered a probiotic, or "good" bacterium, B. subtilis is used to manufacture a range of foods and useful products, including the Japanese delicacy natto, antibiotics, hyaluronic acid used in skincare, and agricultural products to spur plant growth and fight crop diseases.
The cell wall of B. subtilis and other Gram-positive bacteria is made up of two components: peptidoglycan, a layer of sugars and amino acids that is primarily synthesized by clusters of proteins called Rod complexes, and long polymeric molecules called wall teichoic acids. Studies show that when teichoic acids are removed from B. subtilis cells, they lose their rod shape and turn into amorphous blobs. But instead of dying, they grow slowly and stably in this alternate state.
"For decades, we've known that if you get rid of teichoic acids in rod-shaped bacteria, the cells turn into blobs, but we didn't know why," said Enrique Rojas, associate professor of biology at NYU and the study's senior author. "Our study resolves the longstanding question of how teichoic acids promote the rod shape of these bacteria."
To study the role of teichoic acids in the cell wall of bacteria, the researchers used a microscope equipped with a special laser that can track individual molecules. They also used a microscopic plumbing system called microfluidics to trap bacterial cells and remove their teichoic acids while monitoring the motion of the proteins that build the cell wall.
"We developed a way to perform chemistry on the surface of living cells while watching subcellular biology at the same time, a technique we call 'in situ biochemistry,'" said Rojas.
The researchers found that eliminating wall teichoic acids in B. subtilis rapidly arrested Rod complexes and simultaneously unleashed the activity of an enzyme called PBP1, which usually plays a minor role in cell wall synthesis by fixing mistakes made by the Rod complexes. This explained why cells turned into blobs—since Rod complexes reinforce the cell wall along its circumference, essentially girdling the cell into a rod shape, whereas PBP1 synthesizes peptidoglycan in random directions, leading to a blob shape.
The next big question was how teichoic acids controlled the proteins. Since PBP1 was thought to mend holes in the cell wall, the scientists wondered if it was taking over from Rod complexes when teichoic acid depletion exposed pores in the cell wall. To test this, they collaborated with Zarina Akbary, a PhD student in Rojas's lab and study coauthor, who developed a new method to measure the cell wall's porosity with nanoscopic resolution. The team found that nanometer-sized holes appeared within minutes of teichoic acid depletion.
"Our study gets at a fundamental function of teichoic acids: they pave the cell surface of the cell so that Rod complexes don't fall into cell wall potholes and PBP1 doesn't overreact to small defects left by Rod complexes," said Rojas.
Amorphous growth was not only driven by PBP1—it also required the enzyme. In bacteria lacking it, depleting teichoic acids led to cell wall thinning, a dramatic increase in cell wall pores, and a complete arrest of cell growth—all while retaining the bacterium's rod shape. In addition to PBP1, the researchers found that cell growth without teichoic acids also required a second enzyme, LytE, that chops up the cell wall and is needed for rod-shaped growth.
"Our findings reveal that growth without wall teichoic acids is driven by a combination of PBP1 and LytE, meaning that teichoic acids regulate both cell wall synthesis and degradation in B. subtilis. The cells possess a completely different mode of growth driven by these auxiliary enzymes, like a backup plan for when they are treated with drugs that inhibit teichoic acid synthesis," said Barber.
The research provides clues about how the first bacteria on Earth, which probably lacked any well-defined shape, thrived.
"For Bacillus subtilis, amorphous growth requires far fewer proteins than rod-shaped growth. Cells without teichoic acids could therefore represent a model for simpler, primordial cells. The same basic principles that underlie proliferation of teichoic acid-less cells may have been responsible for the proliferation of early blob-like life on Earth," said Barber.
The findings may have implications for many other bacteria besides B. subtilis. For example, Listeria monocytogenes, another rod-shaped Gram-positive bacteria and common culprit in food-borne illness, also loses its shape when teichoic acids are depleted. Studies also show that blocking the synthesis of teichoic acids in methicillin-resistant Staphylococcus aureus (MRSA)—the most notorious antibiotic resistant bacterium—using an FDA-approved antiplatelet drug can re-sensitize it to antibiotics.
This research, co-authored by Zhe Yuan, Jacob Biboy, and Waldemar Vollmer, was supported by the National Science Foundation (2047404) and the National Institutes of Health (R35GM143057).
