This Month in the Blogs (#1?)

In case you missed them:

Merry Youle, at Small Things Considered, writes up a beautiful piece on the direct connections of microbes to the multicellular world. She walks us through some recent work highlighting the connections between parasitic wasps, the aphids they lay eggs within, the bacteria that colonize the aphids, and ultimately to the phage that lysogenize those bacteria. Truly remarkable!

Urinary tract infections are one of the most common infections in the country, costing around $2.4 billion dollars a year. Alan Cann over at MicrobiologyBytes discuss the first ventures into vaccine development for this rather uncomfortable and widespread disease.

I was lucky enough to start my research career by isolating and characterizing a novel bacteriophage of the Mycobacteria. This lead to some interesting findings, including the first description of putative transposons in these particular phages(which we published in this month's Microbiology). Phagehunting, however, is not restricted to only Graham Hatfull's lab, where I did my undergraduate work. The Howard Hughes Medical Institute has funded curriculum development to bring this research experience to colleges across the country. John Dennehy, at The EvilutionaryBiologist, talks about his new Phage Hunting course at Queen's College.

And finally Nick Oswald, at BiteSize Bio, tells us to simmer down when our experiments give us results other than what we expected. Instead, we should concentrate on making sure we are asking the right question.

Enjoy!

And as always, questions, comments, suggestions, etc. are more than appreciated!

The Next Steps in Synthetic Biology

The advent of genome sequencing and analysis, coupled with the relative simplicity of DNA synthesis, has given rise to the field of synthetic biology. Described in 1974 by Waclaw Szybalski, the practice of synthetic biology would include the ability to "devise new control elements and add these new modules to the existing genomes or build up wholly new genomes."

More than 20 years later, this description is being realized. With the great minds and funds of the Venter Institute, we have seen the development of a completely synthetic genome and whole genome transplantation. The genome of Mycoplasma genitalium (at ~580Kbp) was synthesized primarily in vitro before being pieced together in Saccharomyces cereviseae. The whole genome of M. mycoides was transplanted into M. genitalium, changing the metabolism, physiology, structure, and subsequently the species of the recipient.

Now, this group has taken this technology one step further. The synthetic genome they pieced together in yeast, must be isolated and transplanted into a donor cell; thus completing the construction of a synthetic, replicating organism.

In a soon-to-be published article in Science, the group describes a method for modifying the complete bacterial genome while in yeast, and then transferring the modified genome back into the original cell. This process allows bacterial genomes to be modified using the well-described genetic systems in yeast, before being introduced. Thus, new possibilities are open for bacteria that once had little to no genetic tools available.

The authors emphasize this development as it directly relates to Mycoplasma biology. They focus on the fact that Mycoplasma and related species now have an entire repertoire of manipulations available to them, however, the ramifications of this experiment certainly do not escape them and should not escape us.

This is the first example of a bacterial cell being created and engineered completely outside the cell itself. Although the recipient cell began with all the necessary physical components for life, this group synthetically created a new organism to control and change those components. This is the closest we have come to synthesizing an organism from scratch.

Some may say this is playing God; however, it will certainly have a positive impact on the development of biofuels, environmental remediation, and chemical synthesis on an industrial scale.

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Citation:
Lartigue, C., Vashee, S., Algire, M., Chuang, R., Benders, G., Ma, L., Noskov, V., Denisova, E., Gibson, D., Assad-Garcia, N., Alperovich, N., Thomas, D., Merryman, C., Hutchison, C., Smith, H., Venter, J., & Glass, J. (2009). Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast Science DOI: 10.1126/science.1173759

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Other Articles of Interest:
Phage + Metal = Battery?
Free Hydrogen -- Algal Biofuel Prodution

Prokaryotes Can Do Geometry, and Even Have Their Own Protractor

Despite what once was a popular opinion, bacterial cells are not mere sacks of enzymes. Rather, we are discovering that they are highly structured. (Although, this probably should have been expected) Bacterial cells have to know where their poles are located in order to create such structures as E. coli’s chemotactic nose, Caulobacter’s stalk, and polar flagella. Cells must also be able to recognize where their midpoints are located in order to divide or differentiate properly. Furthermore, we are finding that the bacterial chromosome has a distinct physical arrangement, which can ultimately determine gene expression.

In order to attain such organization, bacteria must have ways to pinpoint proteins to these specific locations. One way they may do this is through curvature sensing. As we can easily see, in rod-shaped cells, poles tend to have a larger curvature than the midsection. In some cases, curvature can differ via the direction, not just magnitude. In the case we will look at today, done by the Richard Losick lab, a protein recognizes the positive curvature of a forming forespore.

SpoVM (“Spo-Five-Em”) is a Bacillus protein that localizes to the outer forespore membrane during the beautiful process of sporulation. Despite being a membrane protein produced in the mother cell, SpoVM is present only on the forespore and does not appear at all in the greater cell membrane. Therefore, this raises the obvious question of how such recognition occurs.

The first (and most probable) method is that known as “diffuse and capture.” The idea is that SpoVM localizes to the cell membrane and then diffuses through to the connected membrane of the developing forespore. Once here, a forespore-produced membrane protein captures the diffusing SpoVM. To test this, the Losick lab prevented SpoVM production until after the forespore engulfment. This way, there would be no contiguous membrane from the mother cell to forespore for SpoVM to diffuse through.

The result was that SpoVM still localized only to the forespore, despite a lack of continuous membrane. Since this ruled out diffusion, SpoVM must have another method for such a specific localization. Rather than attempting to address whether a second protein attracts SpoVM (a question that would be slightly difficult to address) the researchers thought that localization might occur due to the specific curvature of the forespore. A logical thought, since the forespore is the only positively curved membrane structure in the Bacillus cell.

If true, then SpoVM should localize to any membrane with positive curvature without regard to the membrane’s original source, location, or other proteins present. In fact, this is what occurs. SpoVM localizes to any positively curved membrane surface. This was shown in vivo with mutant Bacillus that formed internal vesicles, along with mutants of E. coli and Saccharomyces cerevisae that have the same phenotype. Furthermore, in a completely cell free system, SpoVM shows the same localization to small membrane vesicles. Mutants of SpoVM can also be isolated that show a non-discriminatory phenotype and will localize to membranes without regards to their curvature. This shows that the ability to recognize positive membrane curvature is inherent in the protein itself.

Adding to these data, SpoVM appears to have specificity not just for positive membrane curvature, but also for curves with a diameter of 1 micron or less. This is about the same as a Bacillus forespore.

What is the lesson here? Bacteria have unique ways to examine their surroundings and their selves to allow for proper cellular organization. Although membranes with positive curvature within a bacterial cell are rare (photosynthetic vesicles and forespores), negative curvature is obviously not a rare phenomenon. Perhaps similar mechanisms maybe used to identify cellular poles based on degree of negative curvature. There is even a possibility that curvature-sensing molecules are used in eukaryotes to identify various organelles within the cell.

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Citation
Ramamurthi, K., Lecuyer, S., Stone, H., & Losick, R. (2009). Geometric Cue for Protein Localization in a Bacterium Science, 323 (5919), 1354-1357 DOI: 10.1126/science.1169218

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Other Articles of Interest

Throwing the Clutch (Not the Brake) on Bacterial Flagella

Extracellular Membrane Vesicles in Bacteria: Taking Quorum Sensing in a New Direction