Most bacteria protect themselves from environmental and turgor pressure with a rigid cell wall, made up of long glycan strands crosslinked by short peptides. Despite decades of intensive research, the architecture of this peptidoglycan sacculus was debated. We answered this longstanding question, using ECT to show that glycan strands are arranged circumferentially in both Gram-negative and Gram-positive cells. In order for the cell to grow, the sacculus must be cut and new material inserted without compromising its integrity. Current work focuses on understanding this process using ECT, molecular dynamics, and coarse-grained simulations.
Historically, bacterial cells have been classified on the basis of their ability to retain Gram stain. Gram-negative cells typically have two membranes surrounding a thin layer of peptidoglycan. Gram-positive cells have one membrane and a thicker layer of peptidoglycan. We gained a surprising insight into the relationship between these seemingly different architectures from our ECT studies of sporulation. By imaging a rare endospore-forming Gram-negative bacterium, we found that the inner membrane of the mother cell is transformed into the outer membrane of the germinating spore. This interconversion, and the ability of thick peptidoglycan to be transformed into thin (and vice versa), suggests an evolutionary source of the Gram-negative outer membrane and reveals that monoderm and diderm cell plans may not be so different after all.
The cytoskeleton was long viewed as a hallmark of eukaryotic cells. Even after homologues of all the major eukaryotic cytoskeletal elements were found in bacterial genomes, traditional EM methods largely failed to find filaments inside cells. Using ECT, we have found filaments in nearly every bacterial species we have imaged to date!
A few notable examples follow. In one, we discovered novel filaments of a metabolic enzyme. By depolymerizing a filament, the cell can rapidly activate hundreds of enzymes. This appears to have been co-opted to play a secondary role in determining the shape of Caulobacter crescentus cells, offering an intriguing idea for the origin of the bacterial cytoskeleton. We also discovered bacterial tubulins, which form 5-protofilament bundles, suggesting an ancient bacterial origin of a structure long thought to be a eukaryotic invention. We even discovered cytoskeletal proteins unique to bacteria (bactofilins).
ECT has impressive power in revealing what lies inside cells. But it can also show us what is not there. MreB plays an important role in the maintenance of cell shape in many bacteria, but what exactly that role is remains unclear. Fluorescence microscopy suggested that it forms long helical filaments that could globally coordinate peptidoglycan remodeling. We showed, however, that such long filaments are not found in native cells and in at least one case were an artifact of the fluorescently-tagged protein.
Bacteria may not have organelles like eukaryotes, but we are finding more and more cases of sophisticated interior organization. Our lab has described the structures of carboxysomes (used to concentrate RuBisCO), storage granules, magnetosomes, and protein diffusion barriers in the stalk of Caulobacter crescentus. Current work focuses on these and other functional compartments, including the nucleoid, the genome-containing region of the bacterial cell.
Cell division is a fundamental process and we aim to understand it across all three kingdoms of life.
Bacteria and most archaea use FtsZ, an actin homologue, to divide. Our lab was the first to image FtsZ filaments, in Caulobacter crescentus. Rather than a continuous ring, we observed short arcing filaments, supporting a mechanism of constriction based on conformational changes in FtsZ. Debate continues, and current work aims to further elucidate the mechanism of Z-ring contraction. Other archaea, and most eukaryotes, use ESCRT to divide. We imaged dividing Sulfolobus acidocaldarius and found a belt of ESCRT filaments, suggesting a spiraling constriction mechanism.
Motility and Navigation
Single-celled organisms are frequently on the move, and we aim to understand the diverse mechanisms they use to get around. Many bacteria use a complicated rotary motor to spin a long flagellum. Our group has dissected this cellular nanomachine to pseudo-atomic detail and compared it across 11 bacteria. Other bacteria use extendable pili to attach to a surface or to other cells, retracting the pili to pull themselves forward.
Using sub-tomogram averaging of wild-type cells and mutant strains in which each protein component has been knocked out or tagged, we produced a working architectural model of this Type IV pilus that gave insights into how it works.
We also study other bacterial motility mechanisms.
How do cells know which way to go? Chemotaxis has long been a main focus of our research. We combined ECT with X-ray crystallographic structures to generate a pseudo-atomic model of the chemoreceptor array, revealing an extended lattice of interlocking chemoreceptors and associated proteins, supporting the idea that rotation of one ring could drive the rotation of neighboring rings, perhaps explaining the exceptionally high cooperativity of this signaling system. We showed that this architecture is conserved across bacteria and archaea, highlighting its fundamental importance. We also provided insight into the activation of downstream signaling through the release of sequestered kinase subunits. Current work focuses on furthering our understanding of the structural changes underlying signaling.
Competition and Warfare
Single-celled organisms are skilled in the art of war, and we are figuring out their arsenal.
Our lab has revealed the structure of the “spring-loaded molecular daggers” (or Type VI secretion systems) some bacteria use to kill neighboring bacterial cells. We discovered the spectacular “death star” of pyocins that is secreted by a marine bacterium for an as-yet unknown purpose. We also study the Type III secretion system used by pathogenic bacteria.