A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall ...of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8-14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.
The formation of gas vesicles has been investigated in bacteria and haloarchaea for more than 50 years. These air-filled nanostructures allow cells to stay at a certain height optimal for growth in ...their watery environment. Several gvp genes are involved and have been studied in Halobacterium salinarum, cyanobacteria, Bacillus megaterium, and Serratia sp. ATCC39006 in more detail. GvpA and GvpC form the gas vesicle shell, and additional Gvp are required as minor structural proteins, chaperones, an ATP-hydrolyzing enzyme, or as gene regulators. We analyzed the Gvp proteins of Hbt. salinarum with respect to their protein–protein interactions, and developed a model for the formation of these nanostructures. Gas vesicles are also used in biomedical research. Since they scatter waves and produce ultrasound contrast, they could serve as novel contrast agent for ultrasound or magnetic resonance imaging. Additionally, gas vesicles were engineered as acoustic biosensors to determine enzyme activities in cells. These applications are based on modifications of the surface protein GvpC that alter the mechanical properties of the gas vesicles. In addition, gas vesicles have been decorated with GvpC proteins fused to peptides of bacterial or viral pathogens and are used as tools for vaccine development.
Halophilic Archaea (Haloarchaea) thrive in salterns containing sodium chloride concentrations up to saturation. Many Haloarchaea possess genes encoding gas vesicles, but only a few species, such as ...Halobacterium salinarum and Haloferax mediterranei, produce these gas-filled, proteinaceous nanocompartments. Gas vesicles increase the buoyancy of cells and enable them to migrate vertically in the water body to regions with optimal conditions. Their synthesis depends on environmental factors, such as light, oxygen supply, temperature and salt concentration. Fourteen gas vesicle protein (gvp) genes are involved in their formation, and regulation of gvp gene expression occurs at the level of transcription, including the two regulatory proteins, GvpD and GvpE, but also at the level of translation. The gas vesicle wall is solely formed of proteins with the two major components, GvpA and GvpC, and seven additional accessory proteins are also involved. Except for GvpI and GvpH, all of these are required to form the gas permeable wall. The applications of gas vesicles include their use as an antigen presenter for viral or pathogen proteins, but also as a stable ultrasonic reporter for biomedical purposes.
The interactions of the four gas vesicle proteins GvpA, C, N, and O were investigated by split-GFP and pulldown assays. GvpA forms the ribs of the gas vesicle shell, whereas GvpC is attached to the ...exterior surface and stabilizes the gas vesicle structure. The AAA-ATPase GvpN as well as GvpO is found in much lower amounts. GvpN and GvpO formed homodimers and also the GvpN/GvpO heterodimer; both interacted with the C-terminal domain of GvpC when tested by split-GFP. When analyzed by pulldown assays, GvpN and GvpO also selected GvpA. The N-and C-terminal fragments of GvpC dimerized as Cterm/Cterm and Cterm/Nterm, but not as Nterm/Nterm. These interactions at both termini might lead to a network of GvpC molecules at the gas vesicle surface. However, a GvpA/GvpC interaction was not detectable, suggesting that the contact of both proteins is either mediated by another Gvp, or requires different structures that might form when GvpA is aggregated in the gas vesicle shell. Interactions of GvpA, C, N, and O were also studied with the accessory proteins GvpF through GvpM by split-GFP. GvpN bound GvpL only, whereas GvpO interacted with GvpF, I, and L, and the C-terminal domain of GvpC contacted GvpF, H, I, and L. GvpA/GvpA interactions were difficult to detect by split-GFP, but GvpA selected except for GvpI, K, and L all other accessory Gvp in pulldown assays. We will discuss the implications of these findings on gas-vesicle assembly.
The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and ...fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten amino acid substitutions, resulting in mGFP6. Using mGFP6 as reporter, we studied six haloarchaeal promoters exhibiting different promoter strengths. The strongest activity was observed with the housekeeping promoters
of the ferredoxin gene and
of the ribosomal 16S rRNA gene. Much lower activities were determined for the promoters of the p-vac region driving the expression of gas vesicle protein (
) genes in
PHH1. The basal promoter strength dropped in the order
,
>
,
. All promoters showed a growth-dependent activity pattern. The GvpE-induced activities of
and
were high, but lower compared to the
or
promoter activities. The mGFP6 reporter was also used to investigate the regulatory effects of 5'-untranslated regions (5'-UTRs) of three different
mRNAs. A deletion of the 5'-UTR always resulted in an increased expression, implying a negative effect of the 5'-UTRs on translation. Our experiments confirmed mGFP6 as simple, fast and sensitive reporter to study gene expression in haloarchaea.
Halobacterium salinarum
forms gas vesicles consisting of a protein wall surrounding a gas-filled space. The hydrophobic 8-kDa protein GvpA is the major constituent of the ribbed wall, stabilized by ...GvpC at the exterior surface. In addition, eight accessory Gvp proteins are involved, encoded by
gvpFGHIJKLM
that are co-transcribed in early stages of growth. Most of these proteins are essential, but their functions are not yet clear. Here we investigate whether GvpF through GvpM interact. Pull-down experiments performed in
Haloferax volcanii
with the cellulose-binding-domain as tag suggested many interactions, and most of these were supported by the split-GFP analyses. The latter study indicated that GvpL attracted all other accessory Gvp, and the related GvpF bound besides GvpL also GvpG, GvpH and GvpI. A strong interaction was found between GvpH and GvpI. GvpG showed affinity to GvpF and GvpL, whereas GvpJ, GvpK and GvpM bound GvpL only. Using GvpA for similar analyses yielded GvpF as the only interaction partner. The contact site of GvpF was confined to the N-terminal half of GvpA and subsequently mapped to certain amino acids. Taken together, our results support the idea that the accessory Gvp form a complex early in gas-vesicle assembly attracting GvpA
via
GvpF.
R1 is an extremely halophilic archaeon, able to attach to the surface and to form characteristic biofilm structures under physiological conditions. However, the effect of environmental stress factors ...like heavy metals on biofilms was still unknown. Here, we report on the first insights into
biofilm formation when exposed to copper, nickel and zinc and describe the effects of metal ions on the architecture of mature biofilms. We also studied the effects on gene expression in planktonic cells. Investigation of planktonic growth and cell adhesion in the presence of sub-lethal metal concentrations yielded an up to 60% reduced adhesion in case of copper and a significantly enhanced adhesion in case of zinc, whereas nickel treatment had no effect on adhesion. A PMA-qPCR assay was developed to quantify live/dead cells in planktonic cultures and mature biofilms, enabling the investigation of cell vitality after metal exposure. An increased resistance was observed in biofilms with up to 80% in case of copper- and up to 50% in case of zinc exposure compared to planktonic cells. However, nickel-treated biofilms showed no significant increase of cell survival. Microscopic investigation of the architecture of mature biofilms exposed to lethal metal concentrations demonstrated an increased detachment and the formation of large microcolonies after copper treatment, whereas the number of adherent cells increased strongly in nickel-exposed biofilms. In contrast, zinc exposed-biofilms showed no differences compared to the control. Analysis of the expression of genes encoding putative metal transporters by qRT-PCR revealed specific changes upon treatment of the cells with heavy metals. Our results demonstrate diverse effects of heavy metal ions on
and imply a metal-specific protective response of cells in biofilms.
In recent years, synthetic riboswitches have become increasingly important to construct genetic circuits in all three domains of life. In bacteria, synthetic translational riboswitches are often ...employed that modulate gene expression by masking the Shine-Dalgarno (SD) sequence in the absence or presence of a cognate ligand. For (halo-)archaeal translation, a SD sequence is not strictly required. The application of synthetic riboswitches in haloarchaea is therefore limited so far, also because of the molar intracellular salt concentrations found in these microbes. In this study, we applied synthetic theophylline-dependent translational riboswitches in the archaeon
Haloferax volcanii
. The riboswitch variants A through E and E
∗
were chosen since they not only mask the SD sequence but also the AUG start codon by forming a secondary structure in the absence of the ligand theophylline. Upon addition of the ligand, the ribosomal binding site and start codon become accessible for translation initiation. Riboswitch E mediated a dose-dependent, up to threefold activation of the
bgaH
reporter gene expression. Raising the salt concentration of the culture media from 3 to 4 M NaCl resulted in a 12-fold increase in the switching capacity of riboswitch E, and switching activity increased up to 26-fold when the cultivating temperature was reduced from 45 to 30°C. To construct a genetic circuit, riboswitch E was applied to regulate the synthesis of the transcriptional activator GvpE allowing a dose-dependent activation of the
mgfp6
reporter gene under
P
pA
promoter control.
The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of ...mutations in GvpJ and GvpM was studied in
transformants by complementing the respective mutated gene with the remaining
genes and inspecting the cells for the presence of gas vesicles (Vac
). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac
ΔJ + J
transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac
ΔM + M
transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac
phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1-25 and M60-84 fragments of GvpM, and fragment J1-56 of GvpJ interacted with the N-terminal fragment A1-22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other.
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