From 2007.igem.org

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Background: Gas Vesicles: By Phillip Dodson 19/4/2007

In 1998 Maura Cannon and Ning Li expressed gas vesicles from Bacillus Magaterium in E.cloi.[1] The expression was highest with extracted plasmid pNL29 (pBluscriptIIKS expression plasmid) which Maura Cannon has kindly made available to us. The plasmid contains a 6Kb cluster of 11 genes (gvpQ,B,R,N,F,G,L,S,K,J,T,U)[1]. “gvp” stands for gas vesicle protein. In other species eight genes (gvpF,G,J,K,L,M,A,O) have been found to be necessary and sufficient.[2] .

Gas vesicles are present in some halobacteria and most phytoplankton due to their need to float to close to the water surface to collect light. It is expected that they can regulate their position in the water column to balance photo damage with energy generation by photosynthesis, however this is a confounding control system since energy leads to heavy molecule formation which also effects buoyancy.[3, 4]

In all cases the capability is associated with a single gene cluster of about 8-15 genes of gvpA-R. Conservation within the cluster is limited.

The vesicles generated are cylinders with conical ends 50nm to 1um long and 30 to 250nm wide(see figure3). The length is thought to be controlled by gvpI. The width varies between species with some having tight control and others variable morphology. Small maximum diameter results in increased strength and an ability so withstand more rapid changes in static pressure. The width is thought to be controlled by 33AA repeats in gvpC[3]. Details of the proposed biochemical structure can be found in [4], and atomic force microscopy in [5].

Vesicles cluster into groups to form what appear to be much larger gas spaces in cells.[4](see figure 2). The vesicles are mostly made of a single protein gvpA[6], reinforced on the outside by gvpC and gvpH[6]. They form as two opposed and growing cones which grow to a certain diameter after which they extend as cylinders. The cones exclude water but allow free transit of gasses. [4]

Collapsing the vesicles with a rapid change of pressure is irreversible. Care must be taken in centrifuge steps.

The vesicles presence can be observed with x1000, phase contrast microscopy[1] (see figure1), but detailed structure requires TEM [1] and with TEM cryo fracture techniques[4](see figure 3), or TEM platinum-carbon shadow[3]. Deletion of the central part of gvpD resulted in excessive numbers of vesicle in Haloferax voicanni.[4]. In the same species gvpE is the activator of gvpA, gvpD is a repressor of gvpA [7]. The activator and repressor act on four promoter regions pA,pD,pF and pO[8]. gvpC repeats appear to bind gvpA at both ends as shown by trypsin digest [9].

Table 1: Proteins in pLN29 and suspected function by homology.

• Protein Size Function Reference

• gvpB 88 (94 gvpA) main structural protein [6]

• gvpF 255 Nucleation protein (coiled coil)  reduced formation. [10]

• gvpG 88 Present in imunoblot of vessicles [11]

• gvpJ 100 (33% gvpA) change in coil size with development. [4, 10]

• gvpK 94
• gvpL 269 Nucleation protein (coiled coil)  eliminated vesicles. [10]

• gvpN 308 Some similarity to gvpD, thought to regulate vesicle numbers. [4]

• gvpQ? 157 Partially deleted in pLN29
• gvpR 88
• gvpS 95 (30gvpA),(37gvpJ)
• gvpT 292
• gvpU 138

BIBLIOGRAPHY:

• 1. Li, N. and M.C. Cannon, Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli. Journal of Bacteriology, 1998. 180(9): p. 2450-2458.
• 2. Offner, S., et al., Eight of fourteen gvp genes are sufficient for formation of gas vesicles in halophilic archaea. Journal of Bacteriology, 2000. 182(15): p. 4328-4336.
• 3. Dunton, P.G. and A.E. Walsby, The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length. Fems Microbiology Letters, 2005. 247(1): p. 37-43.
• 4. Walsby, A.E., GAS VESICLES. Microbiological Reviews, 1994. 58(1): p. 94-144.
• 5. McMaster, T.J., M.J. Miles, and A.E. Walsby, Direct observation of protein secondary structure in gas vesicles by atomic force microscopy. Biophysical Journal, 1996. 70(5): p. 2432-2436.
• 6. Pfeifer, F., et al., Gas vesicle formation in halophilic archaea. Archives of Microbiology, 1997. 167(5): p. 259-268.
• 7. Pfeifer, F., et al., Regulation of gas vesicle formation in halophilic archaea. Journal of Molecular Microbiology and Biotechnology, 2002. 4(3): p. 175-181.
• 8. Hofacker, A., et al., GvpE- and GvpD-mediated transcription regulation of the p-gvp genes encoding gas vesicles in Halobacterium salinarum. Microbiology-Sgm, 2004. 150: p. 1829-1838.
• 9. Dunton, P.G., et al., Analysis of tryptic digests indicates regions of GvpC that bind to gas vesicles of Anabaena flos-aquae. Microbiology, 2006. 152(Pt 6): p. 1661 - 9.
• 10. Shukla, H.D. and S. DasSarma, Complexity of gas vesicle biogenesis in Halobacterium sp strain NRC-1: Identification of five new proteins. Journal of Bacteriology, 2004. 186(10): p. 3182-3186.
• 11. Becker, S., P.K. Hayes, and A.E. Walsby, Different gvpC length variants are transcribed within single filaments of the cyanobacterium Planktothrix rubescens. Microbiology-Sgm, 2005. 151: p. 59-67.