The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.     

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.     

The accessory gas vesicle protein GvpM of haloarchaea and its interaction partners during gas vesicle formation

Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M_GFP contained a single fluorescent aggregate per cell, whereas cells containing L_GFP or H_GFP were fully fluorescent. The soluble L_GFP formed stable co-aggregates with GvpM in L_GFPM transformants, but the presence of GvpH resulted in the absence of M_GFP foci in HM_GFP transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44–48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac^? transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.     

Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii

Gas vesicles are proteinaceous, gas‐filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in‐silico 3D‐model of GvpA of the predicted coil‐α1‐β1‐β2‐α2‐coil structure is available and implies that the two β‐chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + A_mut transformants for their ability to form gas vesicles (Vac⁺ phenotype). In most cases, an alanine substitution of a non‐polar residue did not abolish gas vesicle formation, but the replacement of single non‐polar by charged residues in β1 or β2 resulted in Vac^– transformants. A replacement of residues near the β‐turn altered the spindle‐shape to a cylindrical morphology of the gas vesicles. Vac^– transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt‐bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac^– transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid‐state NMR.     

Use of cyanobacterial gas vesicles as oxygen carriers in cell culture

The gas vesicles isolated from the cells of filamentous cyanobacterium Anabaena flos-aquae were treated and sterilized with glutaraldehyde and then evaluated for their effectiveness as gas carriers in cell culture. Anchorage-dependent Vero cells were grown in a packed bed of microcarrier beads under the perfusion of Dulbecco’s Modified Eagle’s Medium with 1% serum. The culture medium supplemented with 1.8% (v/v) gas vesicles was found to support a 30% higher maximum glucose utilization rate than the same medium without gas vesicles. The gas vesicle suspension was confirmed to have no apparent effects on cell metabolism in T-flask cultures. The study results indicated that the gas vesicles, with high oxygen carrying capacity, can be used to increase the oxygen supply in cell culture systems.     

A simple and defined method to study calcification by isolated matrix vesicles. Effect of ATP and vesicle phosphatase.

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 microgram of protein, were incubated for 2 h in a 45Ca-labelled solution with 2.2 mM Ca2+, 1.6 mM PO 3/4-and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO4 hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca2+ or Mg2+ was found to stimulate matrix vesicle ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, beta-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, vesicles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.     

A simple and defined method to study calcification by isolated matrix vesicles effect of ATP and vesicle phosphatase

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 μg of protein, were incubated for 2 h in a ⁴⁵Ca-labelled solution with 2.2 mM Ca²⁺, 1.6 mM PO₄^3? and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO₄ hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca²⁺ or Mg²⁺ was found to stimulate matrix vesicle. ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, β-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, veiscles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.     

Gas vesicle assembly in Microcyclus aquaticus.

When observed in the electron microscope intact gas vesicles appeared as transparent areas in whole cells of Microcylus aquaticus, whereas vesicles collapsed by centrifugation were not discernible. Within 5 min of suspending cells containing collapsed vesicles in growth medium, small transparent vesicles were detected. By 15 min the average number of vesicles per cell was 15. This number remained relatively constant while the size of the vesicles increased until they attained their maximum diamtere of 100 nm. At this time the vesicles, interpreted as biconical structures, began to elongate presumably due to the synthesis of the cylindrical midsection. Closely correlated with the time at which vesicles began to elongate was the initiation of smaller vesicles which resulted in a doubling of the number of vesicles per cell by 90 min. This evidence coupled with the isolation of a mutant which assembles only the conical portions of the vesicle suggests that assembly occurs in two distinct stages subject to genetic mutation. Protein and ribonucleic acid synthesis, and presumably adenosine triphosphate formation, were required for gas vesicle assembly. In addition, inhibition of protein or ribonucleic acid synthesis resulted in a loss of extant gas vesicles. Over the time course of our study, deoxyribonucleic acid synthesis was not required for gas vesicle assembly or stability.     

Thyrotropin effects on vesicle transfer and thyroid follicle morphogenesis: a stereological study in the rat

Incubation in a culture medium with and without TSH of 16 day-old foetal thyroid glands induces hypertrophy of the Golgi apparatus which may be correlated with a considerable increase in the number of secretory vesicles. A stereological study performed during the first 6 hr of incubation showed that: vesicle secretion was biphasic; vesicle secretion was heterogeneous with two different populations of vesicles; When TSH (20 mU and 80 mU) was added to the medium, the volume density of the follicular lumina increased; at least during the first 6 hr TSH seemed to be necessary to the formation of follicular lumina.