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.     

Glucose inhibits the formation of gas vesicles in Haloferax volcanii transformants

The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc- gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei . Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing ΔD transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa , gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation.     

The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length

In cyanobacteria the protein on the outside of the gas vesicle, GvpC, is characterised by the presence of a 33 amino acid residue repeat (33RR), which in some genera is highly conserved. The number of 33RRs correlates with the diameter of the gas vesicle and inversely with its strength. Gas vesicles isolated from Microcystis aeruginosa strain PCC 7806 were found to be wider and have a lower critical collapse pressure than those from Microcystis sp. strain BC 8401. The entire gas-vesicle gene cluster of the latter strain was sequenced and compared with the published sequence of the former: the sequences of nine of the ten gvp genes differed by only 1-5% between the two strains; the only substantial difference was in gvpC which in strain BC 8401 lacked a 99-nucleotide section encoding a 33RR. This observation further narrows the correlation of gas vesicle width to the number of 33RRs and suggests how Microcystis strains might be used in experimental manipulation of gas vesicle width and strength.     

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.     

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.     

蓝藻伪空胞的特性及浮力调节机制

伪空胞为蓝藻在水体中提供浮力,使其获得适宜的生长条件,最终导致蓝藻水华暴发,了解伪空胞的特征对控制蓝藻水华暴发有重要意义。文章简要回顾了蓝藻伪空胞自1865年被Klebahn发现到1965年被正式命名的研究历程,目前已发现150多种原核生物中含有伪空胞;伪空胞是两末端呈圆锥状的中空圆柱体,伪空胞半径与临界压强遵循方程:Pc=275(r/nm)-1.67MPa;伪空胞气体含量可根据不同原理,利用Walsby伪空胞测定装置、压力浊度计和细胞流式仪测得。总结了伪空胞组成的化学特性,评述了伪空胞gvp基因丛结构功能和GvpA、GvpC的蛋白空间结构。GvpA是伪空胞合成的主要成分,gvpA在伪空胞内存在多个拷贝,其功能仍不清楚;GvpC由33个氨基酸重复单位组成,重复单位越多,伪空胞越不易破裂;概述了伪空胞3种浮力调节机制:镇重物的改变、伪空胞的合成、伪空胞的破裂;归纳了环境因子(光照、温度、氮、磷、钾)参与伪空胞浮力网络调控的途径。提出了目前伪空胞研究面临的困难和问题,对伪空胞的未来研究方向提出探索性的建议。     

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.     

Isolation of fusion proteins containing SecY and SecE,components of the protein translocation complex from the halophilic archaeon Haloferax volcanii

By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken. Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively. Both fusion proteins were recognized by antibodies raised against the CBD. Thus, CBD-cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE.     

Molecular cloning of A1-ATPase gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The genes encoding A1-ATPase A- and B-subunits were cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the A1-ATPase gene revealed that the A- and B-subunits consisted of 586 and 473 amino acids, respectively. The deduced amino acid sequences of the A- and B-subunits of Ha. japonica showed high identities with those of Halobacterium salinarum and Haloferax volcanii. The consensus ATP-binding motif was found in the A-subunit.     

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.