Team:IISc-Bangalore/Description

  1. Introduction
  2. Structure
  3. Biogenesis
  4. Bioengineering
  5. Purification
  6. References

Gas Vesicles


Figure 1: (left) an algal bloom, (right) micrograph of Microcystis sp. showing gas vesicles

Have you ever seen an algal bloom — a noxious mass of algae polluting eutrophic ponds and lakes? These cyanobacteria, like many other aquatic microorganisms, produce gas vesicles to help them float to the surface. Gas vesicles are hollow gas-filled organelles that make the cell buoyant enough to float in water. The synthesis and degradation of gas vesicles can be controlled by the cell to adjust its vertical position in the water column — a useful ability when competing for sunlight to photosynthesize!

Gas vesicles are ancient organelles, evolving over 3,000,000,000 years ago...


Figure 2: (clockwise from bottom-left) Haloquadratum walsbyi, Nostoc, green sulfur bacteria, Methanosarcina

Gas vesicles are found in a huge diversity of aquatic microorganisms, from green sulfur bacteria and cyanobacteria to methanogens and haloarchaea, indicating an ancient evolutionary lineage stretching back more than three billion years, when these two domains of life — bacteria and archaea — diverged from a common ocean-dwelling ancestor.

Figure 3: Phylogenetic tree showing evolutionary lineage of gas vesicles (green) across the domains of life — bacteria (blue), archaea (red) and eucarya (brown)

Structure of Gas Vesicles

What are gas vesicles made of?

Though “vesicle” conjures up images of phospholipid bilayers, gas vesicles are unique: they are made entirely of protein! Shaped like a cylinder capped by cones, these protein nanostructures consist of “ribs” made of gas vesicle protein A (GvpA), a small, hydrophobic protein that is conserved across all gas-vacuolate species. By forming linear crystalline arrays — the “ribs” — GvpA monomers become the major structural units of gas vesicles.


Figure 4: Electron micrograph of gas vesicles isolated from Anabaena flos-aquae (left) and Halobacterium salinarum NRC-1 (right)

Another gas vesicle protein, GvpC, binds to the gas vesicle surface: by interlocking the GvpA “ribs”, GvpC strengthens the gas vesicles against external pressures. However, gvpC is not an essential part of the gas vesicle gene cluster and several organisms produce gas vesicles without GvpC.

Biogenesis

How are gas vesicles made?

Current evidence suggests that gas vesicle polymerization begins from a bi-cone stage which gradually extends in length to form the cone-capped cylinder. Lengths and widths vary dramatically between species — these dimensions affect the mechanical strength of gas vesicles — but remain fairly constant between cells of the same species. Electron micrography reveals that gas-vacuolate cells contain gas vesicles at all stages of growth.

How are they filled with gas?

Surface tension at the hydrophobic inner surface of the gas vesicle (remember, GvpA is a very hydrophobic protein) excludes water molecules from the interior and only allows the free diffusion of gases through the vesicle walls. As a result, no special mechanism is required to evacuate the gas vesicle; by virtue of their biochemical composition, they fill with gas even as they are synthesized!

Bioengineering Gas Vesicles

How have gas vesicles been modified?

In recent years, labs around the world have bioengineered gas vesicles for diverse purposes — from ultrasonic molecular imaging to vaccine delivery — and this versatility comes from one simple structural feature of the gas vesicle: GvpC. Expressed on the gas vesicle surface, GvpC is a hydrophilic protein that accommodates insertions near its C-terminus, enabling proteins of interest to be fused to it. Antigenic peptides, fluorescent proteins and even enzymes have been successfully expressed on the surface of gas vesicles by fusion to GvpC!

How do we do this?

The in vivo method — demonstrated successfully in Halobacterium salinarum NRC-1 — involves modifying the gvpC gene of gas-vacuolate microbes and purifying modified gas vesicles with the GvpC-fused protein of interest expressed on the surface.

The in vitro method — used on gas vesicles from Anabaena flos-aquae — involves chemically stripping native GvpC off the surface of the gas vesicles, leaving only the GvpA shell, and refolding recombinantly-produced GvpC-fused protein of interest onto the stripped gas vesicle surface.

Purification of Gas Vesicles

Purifying gas vesicles from the microbes which synthesize them is not trivial; even getting intact gas vesicles out of the cells is a painstaking challenge. The first limitation is that gas vesicles have a critical pressure beyond which they collapse irreversibly due to mechanical failure — flattening like a crushed soda can — ruling out cell lysis by sonication or French press. This leaves enzymatic lysis, a far more expensive method that can't easily be scaled up.

There's a more fundamental problem...

Once the cells are lysed and the gas vesicles are released into suspension, another hurdle awaits us — separating the gas vesicles from the remaining cell fractions is not as easy as it sounds. Intuitively, the solution seems obvious: simply allowing the suspension to stand for enough time should make the gas vesicles float to the surface; after all, the gas vesicles have an incredibly low density.

But that does not work.

In fact, the gas vesicle suspension must be centrifuged in a swinging-bucket rotor at very low speeds of ~60 g overnight (to avoid hydrostatic pressure induced collapse) before gas vesicles rise to the surface as a milky-white suspension that must be carefully skimmed off. To ensure sample purity, this tedious process must be serially repeated by diluting the suspension, centrifuging, and skimming off the surface over and over again.

Gas vesicles don't float well in vitro.

For some reason, isolated gas vesicles don't float well at all, unlike gas-vacuolate cells. No bioengineered gas vesicle — despite all its remarkable acoustic, mechanical and surface characteristics — can ever be exploited for its most fundamental property: buoyancy.

And so, for iGEM 2017, we want to make gas vesicles float again — iFLOAT!

References

[1]https://microbewiki.kenyon.edu/index.php/File:CyanobacteriaMicroscope.jpeg
[2]http://www.amyhremleyfoundation.org/images/conservation/html/GasVesicles-07.html
[3]http://www.noaa.gov/what-is-harmful-algal-bloom
[4]http://news.mit.edu/2012/nanotubes-energy-transfer-0706
[5]Walsby AE. Gas vesicles. Microbiological Reviews. 1994;58(1):94-144.
[6]Methanosarcina acetivorans C2A courtesy of Whitehead Institute Center of Genome Research.
[7]http://www.nature.com/news/2004/041011/full/news041011-3.html