"This is both a technological and biological advance", stated Grant Jensen, associate professor of biology at Caltech, a Howard Hughes Medical Institute investigator, and the principal investigator on the study. Their research appears in the on-line early edition of the Proceedings of the National Academy of Sciences (PNAS).
"Bacterial cells rely on a cage-like net that surrounds them to maintain their integrity", Grant Jensen explained. "If it weren't for this molecular bag, the bacteria couldn't survive; they would likely rupture."
This bag, called a sacculus, is made out of peptidoglycan, a mesh-like structure of carbohydrates (glycans) and amino-acid peptides. It is the sacculus, Grant Jensen noted, that is targeted by the antibiotic penicillin; penicillin blocks a bacterium's ability to grow and remodel the bag to fit it as the bacterium itself grows. "If the bug can't make this bag", Grant Jensen stated, "it can't multiply, and you get better."
Researchers have long been interested in understanding the precise architecture of the sacculus. In particular, Grant Jensen and his colleagues have wondered whether the so-called glycan strands - which are cross-linked by peptides to create peptidoglycan - "wrap around the cell like a belt wraps around a person", or whether they stand up from the surface of the bacterial cell, "like grass".
The answer to this debate has eluded the scientists, however, because trying to image such tiny biological objects has been beyond their technological reach. Until now, that is.
"Six years ago, a gift from the Moore Foundation allowed us to buy what is arguably the world's best electron cryomicroscope", stated Grant Jensen. "This allowed us to take a different kind of picture of small biological objects than has ever been possible before. These pictures are 3D images to molecular resolution - you can actually start to see individual biological molecules. Using it, we were able to see this network of glycan strands. It was just remarkable."
By pairing the electron cryotomography and a purification technique that involved removing the sacculi and flattening them in a very thin layer of water, postdoctoral scholar Lu Gan, the paper's first author, was able to image the peptidoglycan structure in three dimensions, which allows for a virtual 3D tour of the bacterial sacculus.
"What we saw were long skinny tubes wrapping around the bag like the ribs of a person or a belt around the waist", stated Grant Jensen. "We also saw that the sacculus is just a single layer thick."
"This is a clear answer to this old question", added Lu Gan. "We now know what the architecture of this most basic shape-determining molecule is. We now know the right answer versus having a family of answers, some of which are wrong."
Understanding how the cell wall is built is important, said Grant Jensen, because scientists have long been in the dark about some of the most basic physical and mechanical aspects of bacterial life, including why they are shaped the way they are. "It's hard to understand how a building is constructed unless you can see the studs", he explained. "Now that we can see the studs - now that we can see the basic architecture of the sacculus - we're closer to understanding how a bacterium could direct its own growth, and how drugs that block that process might work."
Also involved in the research reported in PNAS was Songye Chen, a postdoctoral scholar in biology at Caltech. The paper, "Molecular organization of gram-negative peptidoglycan", was published in the PNAS Early Edition. This work was supported by grants from the National Institutes of Health, a Searle Scholar Award, Caltech's Beckman Institute, and gifts from the Gordon and Betty Moore Foundation and the Agouron Institute. Lu Gan is supported by a fellowship from the Damon Runyon Cancer Research Foundation.