If he casts the right fly, an angler can catch some really big fish. Scientists are the same way, needing the right type of microscope to visualize nature's smallest molecules and atoms. Now, researchers are redesigning their light microscopes to catch a glimpse of some of the most miniscule molecules, those that make proteins in bacteria and archaea.
A promising solution is the use of fluorescence in situ hybridization (FISH) and stochastical optical reconstruction microscopy (STORM). Together, these techniques are improving our understanding of how bacteria and archaea transcribe DNA to RNA and then translate RNA to proteins. In addition, they are re-shaping how cell biology studies relate to environmental microbes.
Luring and Lighting Biomolecules
"Light microscopy has been a workhorse in cell biological research," says Harvard biophysicist Xiaowei Zhuang. She says scientists want to use light microscopy to study cells, especially live ones, because it is non-invasive. The problem, however, with zooming in on biomolecules and their movements in bacteria and archaea is the small size of the individual cells.
At only about three micrometers long and a micrometer wide, bacterial and archaeal cells come into focus just around the diffraction limit of light, which is about 200 nanometers. With light microscopy, scientists can see a cell but not its nuclear and cellular machinery. Even though these cells are relatively simpler than mammalian cells and other eukaryotic ones, scientists still know little about them.
To get a better look, Zhuang and her collaborators developed STORM in 2008 (1). Zhuang's group has used it to image individually labeled proteins in live cells, including bacteria and archaea. And, like pairing the right fly with a great bait, other researchers are using STORM with their own techniques to "look at the distribution and dynamics of nuclear targets at a resolution that is far from the reach of conventional microscopy," says Bakshi.
For example, Cristina Moraru of the Max Planck Institute for Marine Microbiology in Germany and colleagues wanted to know where ribosomes sit within the cell because those molecular machines interact with the nucleoid—the carrier of the genetic information in archaea and bacteria. Based on where ribosomes are located, there are different models of interactions, which can significantly shape regulation of transcription, translation, and other cellular processes.
In a paper recently published in Systematic and Applied Microbiology (2), Moraru’s group reported on a combined STORM and FISH approach to locate ribosomes in an Escherichia coli cell. Moraru’s team used FISH to label specific sequences of ribosomal RNA with fluorescent probes, and then imaged the samples with STORM.
"In the end, all these differences could reflect in the way the cell answer to environmental changes, and therefore, in the fitness and survival," says Moraru. In the near future, she adds, scientists could use STORM, FISH, and other super-resolution techniques to count of the number of ribosomes in a bacterium.
Ribosomal Catch and Release
Counting the number of ribosomes is essential to understanding how bacteria grow. Moraru explains that "the regulation of ribosome numbers in microbial cells is complex and, probably, there will not always be a direct correlation between ribosome numbers and metabolic activity." But it is likely that a cell with a high ribosome content will be more active compared with one with a low ribosome content. If scientists can count ribosomes, they could get a sense of the level of metabolic activity in microbial cells.
But scientists have not yet counted the exact numbers of ribosomes per cell; the FISH protocol and RNA probes need to be more efficient at hybridization. "Work in this direction is in progress, and we are confident that there is only a matter of time till ribosome quantification per cell will be achieved," says Moraru.
So far, prokaryotic cell biology studies have been limited because many methods are not compatible with uncultivated microorganisms. But because the FISH-STORM approach uses RNA probes that target different microbial taxa in environmental samples, scientists could study ribosome variation across bacterial species. "By looking at samples from different environmental conditions, from warm season versus cold season, or, from high salinity versus low salinity, the variation of ribosome number across environmental conditions could be assessed," says Moraru.
In structured environments, such as biofilms, activated sludge and tissue samples, FISH also preserves the spatial information and reveals potential interactions between different species and community members in a sample. "Targeting rRNA by super-resolution FISH is only the beginning. In the near future, we envision targeting the other nucleic acid components of microbial cells to reveal the sub-cellular localization and numbers of specific genes and mRNAs," says Moraru.
A Different Kettle
But the FISH-STORM approach isn't the only way to bait biomolecules in small cells. Bakshi, a graduate student in University of Wisconsin-Madison chemist James Weisshaar's lab, uses a technique called pointillism to do sub-diffraction limit imaging. With this technique, he constructs an image of a cell by localizing a large number of single molecules iteratively. This requires labels that can be switched on and off, but generates resolution up to 20–30 nanometers. In contrast to FISH, Bakshi’s approachcan be used for live-cell imaging.
To truly understand the complexity and heterogeneity of the behavior of any biomolecule, says Bakshi, requires that scientists can probe one molecule at a time. His team's technique gives them the position and movement of a single object in a cell at a high spatio-temporal resolution. "When we are looking at a ribosome, it enables us to determine which molecules are involved in translation and where they are inside the cell," he says.
In a 2012 paper published in Molecular Microbiology (3), he and Weisshaar reported that most of E. coli's translation is not coupled with transcription—a discovery that runs counter to the common view in the scientific literature. Bakshi says that since bacteria lack a nuclear membrane—which separates the nucleoid from the rest of the cytoplasm—co-transcriptional translation is possible in the cells. To what extent the translation process is coupled to transcription, however, was not clear.
Electron microscope images of ribosomes in cell extract, published in the 1970s, suggested that all translating ribosomes are joined to the chromosome through transcriptional coupling. "When we found that our results suggest that most translation is actually happening without such coupling, we were very surprised," says Bakshi. The team eventually figured out that the lifetime of an mRNA in E. coli is much longer than the time taken for its transcription. The mRNA gets released from proteins associated with the nucleoid once transcription terminates and is then translated by ribosomes without being attached to DNA for the rest of its lifetime, he says.
The techniques—whether it's FISH, STORM, or something else—ultimately let biologists cast deeper lines into individual cells of bacteria and archaea, learning more about their molecular and metabolic dynamics.
1. Huang, B., W. Wang, M. Bates, and X. Zhuang. 2008. Three-Dimensional Super-Resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810-813.
2. Moraru, C. and Amann, R. (2012). "Crystal ball: Fluorescence in situ hybridization in the age of super-resolution microscopy." Systematic and Applied Microbiology. In Press.
3. Bakshi, S. et al. (2012). Super-resolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells." Molecular Microbiology 85 (1): 21–38
4. Wang, W. et al. (2011). "Chromosome Organization by a Nucleoid-Associated Protein in Live Bacteria." Science 333: 1445 -1449.