I realize it is a bold statement to compare an iconic superhero to a microscopic bacteria, but how often do you find an organism in real life that can drastically alter its size and shape just due to a small change in its environment? To me, that sounds pretty similar to what happens when the Incredible Hulk gets a little angry. Caulobacter crescentus is a type of bacteria commonly found in freshwater and known for its vibroid (crescent) shape and long stalk. It is a Gram Negative bacteria, meaning it has a cell wall enclosed by two cell membranes, on either side of the wall. It’s most interesting trait of all, in my opinion, is its ability to increase it’s surface area (of both the cell body and stalk) by sevenfold! That would be like you waking up to find your cat is suddenly the size of your golden retriever! This unique capability is deployed when the bacteria finds itself in an environment lacking a key nutrient, phosphate.
The increase in stalk length specifically is beneficial because the stalk is thought to be a critical tool used by the organism to aid in phosphate absorption from the environment. Phosphate is an integral ingredient in making phospholipids. These phospholipids are then used in cell membranes, which are required for the survival of the bacterium. Under normal circumstances, the bacteria would take phosphorus from the environment and use it to construct more cell membrane as it grows. In an environment low in phosphorus, the cell cannot synthesize any new phospholipids needed to support the membrane growth occurring during the excessive cell elongation that C. crescentus undergoes. The question is “what are these bacteria using as the building blocks to construct their new membranes?”. That’s just one of the many questions that Gabriele Stankeviciute and associates pondered during their research on Caulobacter crescentus and phosphate starvation. It has already been shown that almost half of the lipids in C. crescentus’ membrane are glycolipids, an alternative to phospholipids that uses sugar instead of phosphorous, so it is reasonable to guess that C. crescentus might be relying on this other lipid type to build its membrane. The researchers hypothesized that, when phosphate is hard to come by, C. crescentus either makes more of the same glycolipids already in it’s membrane, or it synthesizes a different kind of glycolipid. Using liquid chromatography-mass spectrometry, Stankeviciute and others were able to confirm that both hypotheses were infact occurring during cell elongation. There was a definite increase in the synthesis of the glycolipids already present in the membrane, as well as the production of a new glycolipid. Again, researchers used mass spectrometry, this time in tandem with collision-induced dissociation and mass to identify this new glycolipid as a glycosphingolipid (GSL). Glycosphingolipids are a subset of glycolipids that were previously thought to appear only in eukaryotes and one species of bacteria. The even more exciting news was that the GSL produced by C. crescentus was a previously undiscovered GSL, making it unique to this bacteria. Stankeviciute and associates named this glycosphingolipid GSL-2 because it has two sugar molecules attached to it.
All sphingolipids are synthesized from ceramides, so the process of ceramide production in C. crescentus was an obvious next step to look at in the process of discovering how this bacteria makes its novel GSL. In eukaryotes, ceramide synthesis is a well-known, multistep process, however, in bacteria, the only conserved step between the two groups seems to be one catalyzed by an enzyme called oxoamine synthase. C. crescentus synthesizes three different oxoamine synthases, the genetic codes of which are indicated by the labels CCNNA_01220, CCNNA_01417, and CCNNA_01647. Researchers wanted to know which one(s) were required to make ceramides. The experiment conducted involved wild-type cells (normal, non-mutated cells), a mutant without CCNNA_01220 and another mutant without CCNNA_01647. A CCNNA_01417 mutant was not tested because this enzyme is essential for the cell to stay alive, and scientists are confident that it is not involved in the specific step in question.The three different types of C. crescentus were grown on agar that was low in phosphate to, hopefully, stimulate the production of GSL-2. Next, Stankeviciute and others analyzed the different lipids present in each cell type using mass spectrometry. The results of this experiment can be seen in Figure 4. The wild type had a clear peak visible on the mass spectrometry results that is associated with ceramide. There are a few other peaks on the right of the figure associated with other lipids that the cell is producing. The mutant with a CCNNA_01220 deletion did not have a peak at the ceramide location, meaning that ceramide synthesis was not occurring in those cells. The second mutant lacking CCNNA_01647 was producing ceramide like the wild-type cells. From this, the researchers could conclude that CCNNA_01647 was not needed to synthesize ceramides. Stankeviciute et. al. then went on to restore the deleted gene CCNNA_01220 to the mutant previously lacking it and production of ceramides commenced. From this, they were able to conclude that the oxoamine synthase encoded by CCNNA_01220 is required for the synthesis of ceramides, and thus, the production of GSL-2.
Figure 4: This figure shows the lipid composition of the three different C. crescentus colonies. Notice that the CCNNA_01220 mutant does not have a ceramide peak like the other two. Source.
Following that discovery, Stankeviciute and associates went on to test which genes encoded the enzyme(s) responsible for the next step of GSL-2 synthesis, glycosylation, or the addition of sugars to the ceramide. Because GSL-2 has two sugars, it was suspected that two glycosyltransferases (enzymes that can glycosylate) were needed to add sugars to the ceramides. The results of their experiments showed that two enzymes (Sgt1 and Sgt2) work in sequence, one after the other, to glycosylate the ceramides, and that they are only used by the cell specifically in the glycosylation of sphingolipids.
Stankeviciute et al. did not stop their research regarding GSL-2 in Caulobacter crescentus there. After unlocking some key features of it’s synthesis, the scientists went on to look into other possible roles of GSL-2 and ceramides in the bacterium. They found that a lack of GSL-2 and ceramides did not affect the normal growth of C. crescentus, but they did find that it had a possible role in the bacteria’s defense against antibiotics and phage viruses. GSL-2 and ceramides might not be required for cell life, but they are helpful in ensuring these cells remain alive and even thrive.
The findings of these scientists shed light on a significant portion of the processes involved in phosphate-limited cell elongation in the bacteria Caulobacter crescentus. The research done by these scientists led to the discovery of a novel glycosphingolipid in an organism previously thought not capable of synthesizing one. This research went on to identify not one, but three different enzymes required in the synthesis of GSL-2 (the oxoamine synthase encoded for by CCNNA_01220, Sgt1, and Sgt2). They also began looking into possible roles of GSL-2 and ceramides in antibiotic and phage resistance. I think that this avenue of study could be researched more in depth, and hopefully soon there will be a primary research article dedicated to that aspect alone. Antibiotic resistance is a prevalent area of study. Scientists are constantly battling the ability of some bacteria to become immune to the life-saving antibiotics used to treat the infections they cause. Any research that can shed light on this process could be helpful in giving humans the upperhand against such organisms. While this research might demystify the remarkable, size-changing capabilities of C. crescentus that make it akin to the Incredible Hulk, I think that I gained even more wonder and respect for the bacteria after learning about the mechanisms that go into it’s elongation. I hope you did too!
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