Although visible to the naked eye, microscopy reveals unexpected complexity

At first glance, the slightly cloudy water in the tube looks like a ball of rainwater, with even lighter leaves, debris and threads in the mix. But in the Petri dish, the thin, vermicelli-like threads floating delicately above the leaf debris turn out to be single bacterial cells, visible to the naked eye.

The unusual size is remarkable because bacteria are usually not visible without the aid of a microscope. “It’s 5,000 times bigger than most bacteria. To put it into context, it would be like a human meeting another human as big as Mount Everest,” said Jean-Marie Volland, a scientist with joint appointments at the US Department of Energy (DOE) Joint Genome Institute. (JGI), a DOE Office of Science user facility located at the Lawrence Berkeley National Laboratory (Berkeley Lab) and Complex Systems Research (LRC) Laboratory in Menlo Park, California. In the June 24, 2022 issue of the journal La science, Volland and his colleagues, including researchers from JGI and the Berkeley Lab, LRC, and the University of the Antilles in Guadeloupe, described the morphological and genomic characteristics of this giant filamentous bacterium, as well as its life cycle.

For most bacteria, their DNA floats freely in the cytoplasm of their cells. This newly discovered species of bacteria keeps its DNA more organized. “The big surprise of the project was realizing that these copies of the genome that spread throughout the cell are actually contained within a structure that has a membrane,” Volland said. “And that’s very unexpected for a bacterium. »

Unusual encounters in the Mangroves

The bacterium itself was discovered by Olivier Gros, a professor of marine biology at the University of the Antilles in Guadeloupe, in 2009. Gros’s research focuses on marine mangrove systems, and he was looking for sulfur-oxidizing symbionts in the sulfur-rich mangrove sediments nearby. from his lab when he first encountered the bacteria. “When I saw them I thought, ‘Strange,'” he said. “At first I thought it was just something curious, white filaments that must have been attached to something in the sediment like a sheet. The lab conducted microscopy studies over the next two years and found that it was a sulfur-oxidizing prokaryote.

Silvina Gonzalez-Rizzo, associate professor of molecular biology at the University of the West Indies and co-first author of the study, performed the 16S rRNA gene sequencing to identify and classify the prokaryote. “I thought they were eukaryotes, I didn’t think they were bacteria because they were so big with apparently a lot of filaments,” she recalls her first impression. “We realized they were unique because they looked like a single cell. The fact that they were a “macro” microbe was fascinating! »

“She understood that it was a bacterium belonging to the genus Thiomargarita“, Gros noted. “She named him California. Thiomargarita magnifica. »

“Magnificent because magnus in latin it means big and i think it’s magnificent like the french word gorgeous“, explained Gonzalez-Rizzo. “This type of discovery opens up new questions about bacterial morphotypes that have never been studied before. »

Characterize the giant bacterium

Volland got involved with the giant Thiomargarita bacteria when he returned to the Gros laboratory as a postdoctoral fellow. When he applied for the discovery-based position at the LRC that would see him work at JGI, Gros allowed him to continue his research on the project.

At JGI, Volland began to study California. T. magnifica in Tanja Woyke’s Single Cells Group to better understand what this carbon-fixing, sulfur-oxidizing bacterium was doing in mangroves. “Mangroves and their microbiomes are important ecosystems for the carbon cycle. If you look at the space they occupy globally, it’s less than 1% of the world’s coastal area. But when you then look at carbon storage, you’ll find that they contribute 10-15% of the carbon stored in coastal sediments,” said Woyke, who also leads JGI’s microbial program and is one of the lead authors. of the item. The team was also compelled to study these large bacteria in light of their potential interactions with other microorganisms. “We initiated this project as part of the JGI’s strategic focus of inter-organism interactions, because large sulfur bacteria have been shown to be hotspots for symbionts,” Woyke said. “Yet the project took us in a very different direction,” she added.

Volland took on the challenge of visualizing these giant cells in three dimensions and at relatively high magnification. Using various microscopy techniques, such as hard X-ray tomography, for example, he visualized entire filaments up to 9.66mm in length and confirmed that they were indeed giant single cells rather than of multicellular filaments, as is common in other large sulfur bacteria. He was also able to use the imaging facilities available at Berkeley Lab, such as confocal laser scanning microscopy and transmission electron microscopy (TEM) to visualize cell filaments and membranes in greater detail. These techniques allowed him to observe new membrane-bound compartments that contain clusters of DNA. He dubbed these organelles “pips”, after the small seeds in the fruit. DNA clusters were abundant in individual cells.

The team discovered the genomic complexity of the cell. As Volland noted, “Bacteria contain three times as many genes as most bacteria and hundreds of thousands of genome copies (polyploidy) that spread throughout the cell. The JGI team then used single-cell genomics to analyze five of the bacterial cells at the molecular level. They amplified, sequenced and assembled the genomes. In parallel, Gros’ lab also used a labeling technique known as BONCAT to identify areas involved in protein-making activities, which confirmed that all bacterial cells were active.

“This project has been a great opportunity to demonstrate how complexity has evolved in some of the simplest organisms,” said Shailesh Date, founder and CEO of LRC, and one of the paper’s lead authors. “One of the things that we have argued for is that there is a need to examine and study biological complexity in much more detail than is currently being done. So organisms that we think are very, very simple might have some surprises. »

The LRC funded Volland through grants from the John Templeton Foundation and the Gordon and Betty Moore Foundation. “This groundbreaking discovery underscores the importance of supporting fundamental and creative research projects to advance our understanding of the natural world,” added Sara Bender of the Gordon and Betty Moore Foundation. “We are eager to learn how the characterization of California. Thiomargarita magnifica challenges the current paradigm of what constitutes a bacterial cell and advances microbial research. »

A giant bacterium, several research questions

For the team, characterizing California. Thiomargarita magnifica opened the way to several new research questions. Among them is the role of the bacterium in the mangrove ecosystem. “We know it grows and thrives above the sediments of the mangrove ecosystem in the Caribbean,” Volland said. “In terms of metabolism, it does chemosynthesis, which is a process analogous to photosynthesis for plants. Another open question is whether new organelles called pips played a role in the evolution of the extreme size of Thiomargarita magnifica, and whether or not pips are present in other bacterial species. The precise formation of seeds and how molecular processes inside and outside these structures occur and are regulated also remain to be investigated.

Both Gonzalez-Rizzo and Woyke see successfully growing the bacteria in the lab as a way to get some of the answers. “If we can maintain these bacteria in a lab environment, we can use techniques that aren’t feasible right now,” Woyke said. Big wants to examine other big bacteria. “You can find TEM images and see what the glitches look like, so maybe people saw them but didn’t understand what they were. It will be very interesting to check, if the glitches are already present everywhere. »

Researchers from the National Museum of Natural History (France), Sorbonne University (France) and Cornell University were also involved in this work.