Genetic variation at theapcAB,cpcAB,gvpA1, andnifH loci and in DNA methylation among N2-fixing cyanobacteria designatedNostoc punctiforme

Genetic similarity among cyanobacteria of a morphological subgroup ofNostoc was evaluated through a comparison of several specific genes and the extent of DNA methylation. Four of six cyanobacteria were originally cultured from facultative symbioses with higher plants (Gunnera andEncephalartos); these and one free-living isolate had been identified or reputed to beN. punctiforme. No consistent correlation to species or symbiotic history was found from DNA hybridizations to genes coding for phycocyanin (cpcAB), allophycocyanin (apcAB), gas vesicle protein (gvpA1), and dinitrogenase reductase (nifH). One gene (gvpC) was not present, andgvpA1 was a single-copy gene in all strains. The gas vesicle genes were concluded to be potentially useful for broadly characterizingNostoc or at least this subgroup. Incubations ofNostoc genomic DNA with 22 restriction endonucleases indicated a high degree of methylation and similarity of its methylated DNA to that of other heterocystous cyanobacteria. The genetic variation of theNostoc isolates was judged to reflect primarily different soil origins.     

Gas Vesicle Genes Identified in Bacillus megaterium and Functional Expression in Escherichia coli

Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (gvp) from Bacillus megaterium VT1660 and their functional expression in Escherichia coli. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of E. coli strains that carry this gvp gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-constrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In B. megaterium, the gvp region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and gvpA, -P, -Q, -B, -R, -N, -F, -G, -L, -S, -K, -J, -T, and -U, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in E. coli. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.     

Genetic analysis of the gas vesicle gene cluster in haloarchaea

Gas vesicles are buoyant intracellular organelles composed of a rigid proteinaceous membrane surrounding a gas-filled space. Many prokaryotic microorganisms including photosynthetic and heterotrophic bacteria and halophilic and methanogenic archaea produce gas vesicles. In the majority of cases gas vesicles function in providing vertical motility to cells in aquatic environments. Recent genetic analysis of several halophilic archaeal (haloarchaeal) species has shown that a large cluster of genes [gvpMLKJIHGFEDACN(O)] is necessary for gas vesicle formation.     

Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF–M. The 5′-terminus of the p-gvpF–M mRMA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac^? species Haloferax volcanii as recipient. Construct ΔA containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac^? transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac⁺⁺). Similarly, double transformants containing p-gvpD–M plus p-gvpACNO, or p-gvpG–M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED–ACNO were Vac⁺⁺. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac^?, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF–M plus p-gvpACNO (ΔDE) accumulated gas vesicles (Vac⁺) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF–M mRNA could be suggested for p-gvpD and the 5′- region of its mRNA.     

The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers

A 479-bp bi-directional promoter controls the expression of two genes (mas1′ and mas2′) that encode enzymes for the synthesis of the opine mannopine in plant tissues infected with Agrobacterium tumefaciens. This 5′ regulatory region (mas promoter) contains all the cis-acting elements involved in mediating the complex regulatory properties of these genes in plants. Using different mas promoter regions fused to a minimal 35S promoter (35SΔ108), we found that the regulatory properties of these divergent promoters result from the presence of orientation-dependent negative and positive regulatory regions. Some of these elements have the unusual property of acting as enhancers in one orientation and as silencers in the other. Using electrophoretic mobility shift analysis (EMSA), we showed that the functional mas promoter regions identified by fluorometric and histochemical assays for reporter gene activity in transgenic plants have the ability specifically to bind nuclear protein factors from Nicotiana tabacum, Phaseolus vulgaris, Solanum tuberosum, and Arabidopsis thaliana. Received: 7 May 1999 / Accepted: 5 August 1999     

Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria

Different modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, Serratia sp. ATCC 39006 ( S39006 ). Here we present the first systematic analysis of the genes required to produce gas vesicles in S39006 , identifying how this differs from the archaeon Halobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of S39006 cells, whereas in Escherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in S39006 and E. coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain.     

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.     

Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs<Emphasis Type="Italic"> Glycymeris</Emphasis> sp. (Pteriomorphia) and<Emphasis Type="Italic"> Eurhomalea rufa</Emphasis> (Heterodonta)

Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30–40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae. Electronic Publication     

Gas vesicles are strengthened by the outer-surface protein,GvpC

The critical collapse pressure of gas vesicles isolated from Anabaena flos-aquae decreased from 0.557 to 0.190 MPa when GvpC, the hydrophilic 22 kDa protein present on the outer surface of the gas vesicle, was removed by rising in 6 M urea. Recombinant GvpC was purified from inclusion bodies, produced in an E. coli strain containing an expression vector bearing the gene ecoding GvpC from A. flos-aquae, and then solubilised in 6 M urea. This recombinant GvpC became bound to gas vesicles that had been stripped of their native protein, when the urea was removed by dialysis; the amount which bound increased with the concentration of GvpC present. The critical pressure of these reconstituted gas vesicles increased to 0.533 MPa, 96% of the original value. These results indicate that the function of GvpC is to increase the strength of the structure.Non-standard abbreviations SBTI Soy bean trypsin inhibitor - Gvp Gas vesicle protein - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis     

Analysis of COPII Vesicles Indicates a Role for the Emp47–Ssp120 Complex in Transport of Cell Surface Glycoproteins

Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium‐binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47–Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.      

Distribution, formation and regulation of gas vesicles

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.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Average thickness of the gas vesicle wall in Anabaena flos-aquae.

The average thickness of the layer of protein which forms the wall of the gas vesicles in Anabaena flos-aquae was estimated from measurements of their density and geometry. The volume of the gas space in a purified gas vesicle suspension was determined from the contraction which occurred when the gas vesicles were collapsed by pressure. The volume of the protein in the same sample was calculated from its dry weight and density. From knowledge of the geometry of the average gas vesicle the thickness of the protein layer, 1.54 nm, was then calculated. By a similar method the thickness of the Microcystis gas vesicle wall, 1.62 nm, was calculated from data published by others. The average thickness of the protein layer is, as expected, slightly less than the stacking periodicity of collapsed gas vesicle walls indicated by X-ray diffraction studies.Anabaena gas vesicles with a mean length of 494 nm have an average density of 0.119 mg μl^?1 1 mg of protein is present in gas vesicles having a, total volume of 8.43 μl and a gas space of 7.67 μl. Suspensions of isolated gas vesicles with a gas space concentration of 1 μl ml^?1 give a pressure-sensitive optical density, E_1cm (500 nm) of 2.72, but gas vacuoles in cells give a smaller value.     

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.